Prodrugs of glutamine analogs

ABSTRACT

The disclosure provides compounds having formula (I): 
                         
and the pharmaceutically acceptable salts thereof, wherein R 1 , R 2 , R 2 ′, and X are as defined as set forth in the specification. Compounds having formula (I) are prodrugs that release glutamine analogs, e.g., 6-diazo-5-oxo-L-norleucine (DON). The disclosure also provides compounds having formula (I) for use in treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US2016/044767, filedJul. 29, 2016, that claims the benefit of U.S. Provisional ApplicationNo. 62/199,566, filed Jul. 31, 2015, which is incorporated herein byreference in its entirety.

BACKGROUND

The prodrug approach is a well-established strategy to improvephysicochemical, biopharmaceutic and pharmacokinetic properties ofpotential drug molecules. Approximately 5-7% of drugs approved worldwideare prodrugs with annual sales in 2013 of $11.2 billion. Most prodrugsare simple chemical derivatives of the original molecule. Esterprodrugs, the most common prodrugs, constitute 49% of all marketedprodrugs. Reasons for the popularity of ester prodrugs include theirgenerally straight forward synthesis, their improved lipophilicity andmembrane permeability, and the ubiquitousness of estereases. An exampleof an approach to make an ester prodrug is capping the acidic moiety(ies) with lipophilic alkyl or alkyloxymethyl esters (i.e.,pivaloyloxymethyl (POM) or propyloxy-carbonyloxymethyl (POC); e.g.,Enalapril, Adefovir). Another approach is to cap the acidic moiety(ies)with amino acids to make amides that are recognizable byamidases/peptidases in plasma for hydrolysis or to make them substratesfor transporters, such as Peptide transporter 1 (PEPT1) (e.g.,Pomaglumetad methionil, Valacyclovir).

Glutamine antagonists, such as 6-Diazo-5-oxo-L-norleucine (DON), andaza-serine, have been shown to exhibit broad anti-viral (Antiviral Res.1997; 33(3):165-75; Antiviral Res. 1994; 25(3-4):269-79), anti-infective(J. Bacteriol. 1965; 89:1348-53), anti-cancer (see, e.g., Yoshioka etal., 1992; Tokushima J. Exp. Med. 39(1-2):69-76), anti-inflammatory, andimmunosuppressive activities (Kulcsar et al., 2014; 111:16053-58;Maciolek et al., 2014; Curr Opin Immunol. 27:60-74; Carr et al., 2010; JImmunol. 185:1037-1044; Colombo et al., 2010; Proc Natl Acad Sci USA.107:18868-73), as well as inhibition of convulsions (Proc R Soc Lond BBiol Sci. 1984 Apr. 24; 221(1223):145-68), multiple sclerosis (Tohoku,J. Exp. Med. 2009; 217(2):87-92), epilepsy, and viral encephalitis (J.Neurovirol. 2015 April; 21(2):159-73. doi: 10.1007/s13365-015-0314-6),in many published preclinical and several clinical studies. However, theoccurrence of severe toxicity (e.g., dose limiting GI toxicities, suchas oral mucosistis, gastric bleeding, nausea and vomiting, abdominalpain, leukopenia, thrombocytopenia, and the like) when administeringsuch glutamine antagonists at therapeutic dose levels has hampered theirclinical development.

Prior attempts to mitigate the severe toxicity associated with glutamineantagonists, such as DON, have been unsuccessful. For example, dividingdaily dosing and administering of DON every four to six hours apparentlydoubled DON's toxicity potential (MgGill, et al., 1957). In anotherexample, development of a treatment involving DON dosed with glutaminaseto decrease plasma glutamine so that the DON dose could be reduced washalted after publication of a clinical trial.

SUMMARY

The presently disclosed subject matter provides prodrugs of glutamineantagonists, and pharmaceutically acceptable salts and esters thereof.In some aspects, the presently disclosed subject matter provides aprodrug of a glutamine antagonist, or a pharmaceutically acceptablesalt, the prodrug having a structure of formula (I):

wherein: X is selected from the group consisting of a bond, —O—, and—(CH₂)_(n)—, wherein n is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, and 8; R₁ is selected from the group consistingof H and a first prodrug-forming moiety capable of forming a salt or anester; and R₂ is H or a second prodrug-forming moiety capable of formingan amide linkage, a carbamate linkage, a phosphoramidate linkage or aphosphorodiamidate linkage with the nitrogen adjacent to R₂; R₂′ isselected from the group consisting of H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, or R₂ and R₂′ together form a ring structure comprising—C(═O)-G-C(═O)—, wherein G is selected from the group consisting ofC₁-C₈ alkylene, C₁-C₈ heteroalkylene, C₅-C₈ cycloalkylene, C₆-C₁₂arylene, C₅-C₁₄ heteroarylene, bivalent C₄-C₁₀ heterocycle, each ofwhich can be optionally substituted; or R₁ and R₂′ together form a 4- to6-membered heterocylic ring comprising the oxygen atom adjacent to R₁and the nitrogen atom adjacent to R₂′; provided that the compound has atleast one prodrug-forming moiety selected from the group consisting ofthe first and the second prodrug-forming moieties.

In some aspects, the presently disclosed subject matter provides aprodrug of a glutamine antagonist, or a pharmaceutically acceptablesalt, the prodrug having a structure of formula (IIA) or formula (IIB):

wherein:

R1 is selected from the group consisting of H and C1-6 alkyl;

R11 is selected from the group consisting of H, methyl, isopropyl,sec-butyl, benzyl, p-hydroxybenzyl —CH2CH(CH3)2, —CH2OH,—CH(OH)CH3,—CH2-3-indoyl, —CH2COOH, —CH2CH2COOH, —CH2C(O)NH2,—CH2CH2C(O)NH2, —CH2SH, —CH2CH2SCH3, —(CH2)4NH2, —(CH2)3NHC(═NH)NH2, and—CH2-3-imidazoyl;

R12 is selected from the group consisting of H, C1-4 alkyl, and—C(═O)R13; and

R13 is C1-4 alkyl.

In some aspects, the presently disclosed subject matter provides aprodrug of a glutamine antagonist, or a pharmaceutically acceptablesalt, the prodrug having a structure of formula (III):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₃ and R₄ are independently selected from the group consisting of H,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, aryl, and substituted aryl; and

R₁₀ is C₁₋₆ alkyl.

In other aspects, the presently disclosed subject matter provides apharmaceutical composition comprising a compound of any one of formula(I), formula (IIA), formula (IIB), or formula (III), and apharmaceutically acceptable carrier, diluent, or excipient.

In certain aspects, the presently disclosed subject matter provides amethod for treating a disease or a condition, the method comprisingadministering to a subject in need of treatment thereof, a compound ofany one of formula (I), formula (IIA), formula (IIB), or formula (III),or a pharmaceutical composition thereof, in an amount effective fortreating the disease or condition. In still other aspects, the presentlydisclosed subject matter provides the use of a compound of any one offormula (I), formula (IIA), formula (IIB), or formula (III), or apharmaceutical composition thereof, for treating a disease or condition.In some embodiments, the disease or condition is selected from the groupconsisting of an infection, cancer, an autoimmune disease, aninflammatory disease, and a neurodegenerative or neurological disease.

In yet another aspect, the presently disclosed subject matter provides acompound of any one of formula (I), formula (IIA), formula (IIB), orformula (III), or a pharmaceutically composition thereof, for use as amedicament.

In yet another aspect, the presently disclosed subject matter provides acompound of any one of formula (I), formula (IIA), formula (IIB), orformula (III), or a pharmaceutically composition thereof, for use in thetreatment of a disease or condition, preferably the disease or conditionis selected from the group consisting of an infection, cancer, anautoimmune disease, an inflammatory disease, and a neurodegenerative orneurological disease.

In yet another aspect, the presently disclosed subject matter provides acompound of any one of formula (I), formula (IIA), formula (IIB), orformula (III), or a pharmaceutically composition thereof, for use in thetreatment of the excess and/or aberrant glutamine activity.

Applicant has found that compounds of the disclosure having formula (I),formula (IIA), formula (IIB), and formula (III) are stable in plasma,liver microsomes, liver tissue, and gastrointestinal tissue, yet thesecompounds are cleaved in tumor cells to liberate DON in tumor tissue.The unexpected tumor-targeted properties of compounds having formula(I), formula (IIA), formula (IIB), and formula (III) result in asurprising improvement in therapeutic index for treating cancer with DONand provide the maximum therapeutic benefit to a subject in need of suchtreatment.

Applicant has also found unexpectedly that compounds of the disclosurehaving formula (I), formula (IIA), formula (IIB), and formula (III)exhibit unexpected enhanced CSF to plasma partitioning afteradministration, making them uniquely useful for the treatment of CNScancers such as glioblastoma, oligodendroglioma, neuroblastoma,meningioma, spinal cord tumor and metastatic cancer that has spread tothe central nervous system (CNS).

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 is an illustration showing exemplary amino (R) and carboxy (R′)modifications made to DON in an attempt to synthesize various slowrelease DON prodrugs.

FIG. 2A and FIG. 2B are illustrations demonstrating the chemicalstability challenges encountered when attempting to synthesize DONprodrugs, including stability issues presented by DON esters (FIG. 2A),and DON compounds possessing a free terminal carboxylate (FIG. 2B).

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D are bar graphs demonstrating thefailed chemistry and poor pharmacokinetics of certain attempts to designDON prodrugs. FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show that most DONprodrugs having a free carboxylate group exhibited negligible exposurecompared to DON (except for 26, which showed some release; FIG. 3A) whenadministered orally in mice, including 29 (FIG. 3B), 23 (FIG. 3C) and 30(FIG. 3D), which showed little DON release in mouse plasma upon oraladministration. Note that release of DON prodrug LTP073 was below thelimit of quantitation in all mouse plasma samples at 30 minutes and 5hours after dosing (not shown).

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D are line graphs showingpercentages of DON released in plasma after 0.5 h and 5 h followingadministration of exemplary DON-releasing as compared to DON. FIG. 4Aand FIG. 4B show the results of a pharmacokinetic study in mice showingplasma DON concentrations following administration of DON (1.6 mg/kgp.o.) and DON prodrugs dosed at 1.6 mg/kg equivalent of DON, such as 22(FIG. 4A; 2.5 mg/kg p.o.) and 25 (FIG. 4B; 2.9 mg/kg p.o.). Compound 22showed lesser release at 0.5 h (65% of DON), however, the levels weresimilar at 5 h (almost 100%) to that of DON. FIG. 4C and FIG. 4D showthe results of a pharmacokinetic study in mice showing plasma DONconcentrations following administration of DON (1.6 mg/kg i.p.) and DONprodrugs 17 (FIG. 4C; 3.5 mg/kg p.o.) and 6 (FIG. 4D; 3.7 mg/kg p.o.).

FIG. 5 is a bar graph showing the stability of DON prodrug 25 inphosphate-buffered saline (PBS) and simulated gastric fluid (SGF) after30 min and 60 min.

FIG. 6 is a bar graph showing the results of an in vitro metabolicstability screen of DON prodrug 25 in mouse, monkey, pig, and humanplasma after 30 min and 60 min. 25 was found to be unstable in plasma inall tested species as both the ethyl ester and leucine ester werehydrolysed by plasma esterases.

FIG. 7 is an illustration showing the generic structure of certain celltargeting DON prodrugs that comprise an isopropyl ester carboxymodification.

FIG. 8 is a bar graph showing the results of an in vitro metabolicstability screen of cell targeted phosphamide DON prodrug 4 in mouse,monkey, pig, and human plasma after 30 min and 60 min. Compound 4 wasfound to be unstable in mouse plasma, but stable in the plasma of allother tested species.

FIG. 9 is a bar graph showing the results of an in vitro metabolicstability screen of DON prodrug 7 in mouse, monkey, pig, and humanplasma after 30 min and 60 min. Compound 7 was found to be unstable inmouse plasma, but stable in plasma of all other tested species.

FIG. 10 is a bar graph showing the results of an in vitro metabolicstability screen of DON prodrug 9 in dog, mouse, monkey, pig, and humanplasma after 30 min and 60 min. Compound 9 was found to be unstable inplasma off all tested species. Metabolic identification suggestedhydrolysis of leucine, but stable isopropyl ester for specific releaseinside lymphoid cells.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E and FIG. 11F are bargraphs demonstrating the results of an ex vivo study comparing theaccumulation of DON and DON prodrugs in plasma, blood cells, red bloodcells (RBC), and peripheral blood mononuclear cells (PBMC) after 30minutes of incubation of 20 μM of DON (FIG. 11A) and DON prodrugs 4(FIG. 11B), 7 (FIG. 11C), 9 (FIG. 11D), 13 (FIG. 11E) and 14 (FIG. 11F)in whole human blood;

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D are bar graphs demonstratingthe results of ex vivo studies comparing the accumulation of DON and DONprodrugs in Cynomolgous monkey plasma, blood cells, RBC and PBMC after30 minutes of incubation of 20 μM DON (FIG. 12A) and DON prodrugs,including 20 μM 4 (FIG. 12B), 7 (FIG. 12C) and 9 (FIG. 12C), in monkeywhole blood samples.

FIG. 13A, FIG. 13B, and FIG. 13C are bar graphs demonstrating theresults of ex vivo studies comparing the accumulation of DON and DONprodrugs in pig plasma, blood cells, RBC and PBMC after 30 minutes ofincubation of 20 μM DON (FIG. 13A) and DON prodrugs, including 20 μM 7(FIG. 13B) and 9 (FIG. 13C), in pig whole blood samples.

FIG. 14 is a line graph showing that daily high dose treatment leads toweight loss in DON treated mice. No weight loss was observed in 25treated mice.

FIG. 15 is a table showing that daily high dose treatment leads topan-cytopeniain DON treated mice. Mice treated with 25 exhibited someleukopenia.

FIG. 16 is a table showing that renal and liver function in mice treatedwith either high dose DON or 25 was unaffected.

FIG. 17A, and FIG. 17B are line graphs showing that 25 and DON treatmentcure lymphoma; DON mice die from treatment with DON while 25 is welltolerated.

FIG. 18A and FIG. 18B are bar graphs and FIG. 18C is an illustrationshowing that compound 25 (FIG. 18A) reduces C_(max); (FIG. 18B) enhancestumor to gut ratio; and (FIG. 18C) causes less GI toxicity in mice vsDON at equimolar doses in mice. Ova vaccinia virus infection was givenfollowed by daily treatment with vehicle (Veh), DON (0.8 mg/kg), and 25(0.8 mg/kg equivalent) for 5 days. At 1 hr after last dose, animals weresacrificed, small intestines removed, perfused with DPBS pH 7.4, androlled longitudinally using the “swiss role” technique, and fixative forparaffin wax embedding. Upper panels show hematoxylin and eosin (H&E)stained sections of jejunum-ileum at 4× magnification; lower panel showssame sections at 10× magnification. DON caused significant inflammation,consisting of multifocal crypt distortion (left circle on the bottomleft picture) and dilatation with loss of columnar epithelial morphology(rightcircle circle on the bottom left picture). DON prodrug looked moresimilar to vehicle-treated mice, with slightly hypercellular crypts, butno distortion (left circle on the bottom right picture) and normalcolumnar morphology (right circle on the bottom right picture).

FIG. 19A and FIG. 19B are tables showing that no bone marrow suppressionwas observed (FIG. 19A) following 25 at two 14-day effective dosingschemes; (FIG. 19B) employing long term 25 dosing schemes (48 days).

FIG. 20 is an illustration showing exemplary structures of DON andDON-based prodrugs.

FIG. 21A is a bar graph and FIG. 21B is a line graph showing that DON(1) inhibits glutamine metabolism and glioblastoma multiform (GBM) tumorgrowth in vivo. FIG. 21A shows compound DON (1) (0.8 mg/kg, i.p.)inhibited glutamine metabolism as evidenced by increased endogenousglutamine concentrations in flank GBM tumors 2 hours post-administrationrelative to vehicle-treated controls; *p<0.05. FIG. 21B shows inefficacy studies, compared to Day 0 baseline, vehicle-treated miceexhibited significant growth of flank GBM tumors during the course ofthe experiment. By contrast, systemic administration of 1 (0.8 mg/kg,i.p, q.d. days 1-6) caused a dramatic reduction in tumor size;***p<0.001, ****p<0.0001.

FIG. 22A and FIG. 22B are line graphs and FIG. 22C is a table showing invivo brain and plasma pharmacokinetics of compound DON (1) followingoral administration of DON (1) and 14b in mice. 1 and 14b were dosed inmice at 0.8 mg/kg equivalent, via oral gavage and plasma and brainconcentrations of compound 1 were evaluated via LC/MS. Oraladministration of compound 1 and 14b exhibited similar plasma and brainpharmacokinetic profiles due to complete and rapid metabolism of 14b to1 in mouse plasma.

FIG. 23A is a bar graph, FIG. 23B is a bar graph, and FIG. 23C is atable showing in vivo pharmacokinetics of DON following intravenous(i.v.) administration of DON (1) and 14 in monkey plasma andcerebrospinal fluid (CSF). 1 and 14b were dosed in two pigtail macaquesat 1.6 mg/kg equivalent of 1 via i.v. administration and plasma (0.25-6h) and CSF (30 min) concentrations of DON were evaluated via LC/MS.Relative to 1, 14b delivered substantially lower DON plasmaconcentration. The reverse was observed in CSF, where 14b deliveredsignificantly higher DON CSF concentrations, achieving a 10-foldenhanced CSF to plasma ratio at 30 minute post dose.

FIG. 24 is a bar graph showing species specific plasma stability of 14b;14b is stable in plasma of human, pig, dog and monkeys, but rapidlymetabolized in mice;

FIG. 25 is an illustration showing exemplary structures of DON andDON-based prodrugs 25, 9, 38 and 60; different N-amino acid promoeities(e.g., leucine, tryptophan) provide differential plasma and microsomalstability.

FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 26D are bar graphs showing invitro plasma stability of DON prodrugs 9, 25, 38 and 60. Metabolismoccurs via removal of the N-protecting group; both ethyl and isopropylesters are stable in plasma of pigs and humans.

FIG. 27A, FIG. 27B, FIG. 27C, and FIG. 27D are bar graphs showing invitro liver microsomal stability of DON prodrugs 9, 25, 38 and 60; allprodrugs showed moderate-high stability in human and pig microsomes.

FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D, FIG. 28E, FIG. 28F, FIG. 28G,FIG. 28H, FIG. 28I, and FIG. 28J are bar graphs showing ex-vivo studiesin whole human and pig blood of 9, 25, 38 and 60; DON prodrugsselectively deliver DON to PBMCs in both humans and pigs vs plasma;compared to DON, the PBMC/plasma ratio was enhanced 10-100+ fold;

FIG. 29A FIG. 29B, FIG. 29C, FIG. 29D and FIG. 29E are line graphsshowing pig in vivo studies with DON prodrugs of 9, 25, 38 and 60; DONprodrugs selectively deliver DON to PBMCs vs plasma; compared to DON,the PBMC/plasma ratio was enhanced 6- to 10-fold.

FIG. 30A, FIG. 30B, and FIG. 30C are bar graphs showing plasma stabilityof compound Methyl-POM 14b and its derivatives.

FIG. 31 is an illustration showing exemplary structures ofN-acylalkyloxy DON-based prodrug analogs for intracellular targeting andbrain penetration; the addition of steric bulk to the “bridge” mightresult in a slower hydrolysis.

FIG. 32A, FIG. 32B, and FIG. 32C are line graphs showing pig i.v.studies with DON prodrugs: in vivo in pigs DON prodrugs selectivelydeliver DON to PBMCs vs DON.

FIG. 33 is a bar graph and two tables showing PBMC to plasma AUC(_(0-t))ratio comparison DON prodrugs achieve >6 fold better ratio (based onAUC_(0-t)) vs DON.

FIG. 34A is a line graph and FIG. 34B is a bar graph showing monkey IVstudies with DON prodrug 14b shows lower plasma but higher CSFconcentrations achieving ˜6-7 fold>CSF/plasma ratio vs DON.

FIG. 35 is a bar graph showing CSF to plasma ratio at 1 h after i.v.infusion with compounds 1, 9, 14b, 25 and 60.

FIG. 36 is an illustration showing clinical findings for DON (1) and 38over 5 days observation period DON-treated pigs exhibited more adverseclinical signs versus 38; DON (1.6 mg/kg) and 38 (1.5 mg/kg) doses werechosen to provide equivalent PBMC exposure; drugs were dosed i.v. for 2h/day for 5 days (n=2 pigs); close clinical monitoring was performed toevaluate drug toxicity (e.g. lethargy, anorexia, diarrhea, GI-bleeding,etc); after 5 days of dosing, pigs were euthanized and tissues wereharvested for PK and histopathology.

FIG. 37A, FIG. 37B, FIG. 37C, and FIG. 37D are illustrations showingthat macroscopically, DON-treated pigs exhibited more severe stomachtoxicity versus 38; histopathologic toxicity scoring forgastro-intestinal and other tissues is pending; and

FIG. 38 is a bar graph showing that at equivalent PBMC exposures, 38administration delivers less DON to GI tissues versus DONadministration; DON administration appeared to cause more adverseclinical effects versus 38.

FIG. 39 is a line graph illustrating that administration of DON markedlyinhibits lymphoma growth in a EL4 mouse lymphoma model.

FIG. 40 is a bar graph illustrating that administration of DON has amodest effect on inhibiting melanoma growth, which is a not a bonemarrow derived tumor.

FIG. 41A is a Kaplan-Meier graph and FIGS. 41B-E are line graphs showingthat compound 25 (5 day dosing starting on day 7) is superior to CB-839(a glutamase inhibitor) (30 day dosing starting day 1) in CT26 tumormodel. These unexpected results suggest that 25 can be used as a singleagent to treat colon cancer.

FIG. 42 is a line graph illustrating that compound 25 (4 days startingon day 6) is superior to CB-839 (continuous twice daily dosing startingon day 1 prior to engraftment) in a CT26 tumor model. FIG. 42 shows micereceived daily compound 25 (1.9 mg/kg) on days 6-9 vs BID CB-839 on days1-15. These results again demonstrate the compound 25 is superior toCB-839 and unexpectedly that compound 25 can be used as a single agentto treat cancers such as colon cancer.

FIG. 43 is a line graph illustrating that compound 25 (daily days 7-22)is superior to CB-839 (continuous twice daily dosing days 1-29) in a 4T1breast cancer model. Mice received daily compound 25 (1.0 mg·kg/d) fordays 7-22 as compared to BID CB-839 for days 1-29. These results againdemonstrate the compound 25 is superior to glutamase inhibitor CB-839and unexpectedly that compound 25 can be used as a single agent to treatcancers such as breast cancer.

FIGS. 44A-D and F are line graphs and FIG. 44E is a Kaplan-Meier graphillustrating that compound 25 dosing of 1 mg/kg following by 0.3 mg/kgleads to a complete and durable response in the MC38 tumor model. Theseresults again demonstrate the compound 25 unexpectedly that compound 25can be used as a single agent to treat cancers such as colon cancer

FIGS. 45A, B, and D are line graphs and FIG. 45C is a Kaplan-Meier graphillustrating that compound 25 gives a robust response and improvedoverall survival as compared to vehicle-treated animals in the CT26colon cancer model.

FIGS. 46A, B, and D are line graphs and FIG. 46C is a bar graphillustrating that compound 25 provides a robust response and improvedoverall survival the 4T1 breast cancer model.

FIGS. 47A-47G are line graphs illustrating that mice treated withcompound 25 when administered as a single agent prevents colon cancercell proliferation upon rechallenge, demonstrating that compound 25unexpectedly immunostimulates the mouse.

FIGS. 48A and 48C are line graphs and FIGS. 48B and 48D are Kaplan-Meiergraphs showing that compound 25 inhibits colon cell proliferation andextends the survival of mice in the MC38 model.

FIG. 49A is an illustration of mice and FIG. 49B is a line graphillustrating that compound inhibits tumor growth. FIG. 49A shows miceafter 30 days inoculation (8 days drug free). FIG. 49B shows tumor areavs. days post injection with 4T1 tumor cells. Mice were treated withcompound 25 1 mg/kg every day (from d5-d22) or vehicle. 4T1 tumor cells(0.1 million) were injected into the mammary fat pads of 8-week-oldfemale BALB/c mice. Mice received vehicle (PBS) or 1 mg/kg compound 25daily from day 5 to day 22. Photos were taken on day 30 after tumorinoculation (FIG. 57A). Tumor volume was measured 2-3 times weekly untilWT mice were sacrificed (when size reached to 20 mm length or necrosisoccurred). Day 0: 100K 4T1 cells s.c. in 4th mammary pad. Day 5-22:Daily compound 25. The results show that compound 25 inhibited thegrowth of mammary carcinoma tumor cells.

FIG. 50A is an illustration of mice and FIG. 50B is a bar graphillustrating that glutamine analogue (6-diazo-5-oxo-L-norleucine (L-DON)derivative compound 25 referred to in FIGS. 50A and 50B as “JHU”)inhibits tumor growth. 4T1-Luc tumor cells (0.1 million) were injectedinto the mammary fat pads of 8-week-old female BALB/c mice. Micereceived vehicle (PBS) or 1 mg/kg compound 25 daily from day 7. Anti-PD1(5 mg/Kg) was administered on day 5, 8, and 12. Mice carrying 4T1-luctumors are injected with Luciferin to measure luminescence from thetumor. IVIS imaging were taken on day 13.

FIG. 51A is a line graph and FIG. 51B is a bar graph illustrating thatcompound 25 inhibits tumor growth. FIG. 51B shows tumor weight (mg) onharvest day 21. 4T1-Luc tumor cells (0.1 million) were injected into themammary fat pads of 8-week-old female BALB/c mice. Mice received vehicle(PBS) or 1 mg/kg compound 25 daily from day 7 to day 16. Anti-PD1 (5mg/Kg) was administered on day 5, 8, 12 and 17. Tumor volume wasmeasured 2-3 times weekly until tumors were evaluated for tumorinfiltrating cells on day 21. Tumor weights were measured on day 21. Onday 21, the PD1 group tumor size looks like it was reduced, however, theresult was not due to a tumor size reduction, but rather because one ofthe large tumor mice died.

FIG. 52 is a line graph showing that compound 25 is efficacious in theMC38 tumor model. These data show unexpectedly that compound 25 ishighly effective at killing colon cancer cells as a single agent.

FIG. 53 is a line graph showing compound 25 and DON are efficacious inthe EL4 mouse lymphoma model. These data show unexpectedly that compound25 is highly effective at killing lymphoma cancer cells as a singleagent.

FIG. 54 is a Kaplan-Meier graph showing that DON treated mice die oftoxicity, and compound 25 treated mice show no signs of toxicity in theEL4 mouse lymphoma model. These results show unexpectedly that compound25 is highly effective at killing cancer cells but with significantlyimproved tolerability compared to DON.

FIG. 55 a line graph showing compound 25 administered as a single agentis more efficacious than oxaliplatin in the MC38 tumor model. Theseresults show unexpectedly that compound 25 (referred to as “JHU”) ishighly effective at killing colon cancer cells as a single agent.

FIGS. 56A-C are bar graphs showing compound 60 preferentially deliversDON to P493 tumor cells versus plasma.

FIG. 57 is a line graph showing subcutaneous administration of compound60 unexpectedly promoted tumor regression in the MC38 tumor model as asingle agent.

FIG. 58A is a line graph demonstrating different DON plasma profiles inMonkey for DON and compound 14b.

FIG. 58B is a bar graph showing that compound 14b exhibited enhancedCSF:plasma ratio of DON in Monkey.

FIG. 59A is a line graph demonstrating different DON plasma profiles inswine for DON, compound 14b and compound 47.

FIG. 59B is a bar graph showing that compounds 14b and 47 exhibitedenhanced CSF delivery of DON at 60 min post-administration in swine

FIG. 59C is a bar graph showing that compounds 14b and 47 exhibitedenhanced CSF:plasma ratio of DON at 60 min post-administration in swine.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

I. Prodrugs of Glutamine Antagonists

DON is an antibiotic that was isolated from Streptomyces in 1953. DON isa diazo analog of L-glutamine, which interferes in various reactions inthe synthesis of nucleic acids and proteins in which L-glutamine donatesnitrogen, blocks various glutamine recognizing enzymes, such asglutaminase, modulates brain glutamate levels, and is involved in energymetabolism, amongst others.

One strategy to improve the therapeutic index of DON for varioustreatment regimes, including GBM therapy, would be to increase its brainexposure while limiting its systemic exposure and thus toxicity(Upadhyay, 2014). The prodrug approach is a well-established strategy toalter the pharmacokinetic and tissue distribution of drug molecules,however synthetically this approach is challenging with DON. Given thatDON's labile diazo group is critical to its glutamine antagonizingactivity, the addition of promoeities must be performed under mildconditions to preserve the diazo ketone group.

The presently disclosed subject matter provides novel compositions ofmatter wherein pro-moieties have been added to glutamine antagonists,such as 6-diazo-5-oxo-norleucine (DON), and aza-serine.

The presently disclosed prodrugs of glutamine antagonists were preparedby masking the amine and/or the carboxylate functionalities to alter thepharmacokinetics of DON providing slower release kinetics and cellulartargeting to enhance tolerability. Further, the presently disclosedprodrugs, in some embodiments, selectively target the active glutamineantagonists to specific cells or provide a slower release of DON andthus decrease the toxicity of the drug molecule.

The presently disclosed subject matter demonstrates that masking boththe α-amino group and the carboxy-functionality to be derivatizedenhances prodrug stability and oral bioavailability. The presentlydisclosed prodrugs also exhibit a stability that is comparable to freeDON.

Structures of representative DON prodrugs are provided in Table 1.

TABLE 1 Structures of Representative DON Prodrugs IOCB No./ Compound No.Structure MW Compound 1  (DON)

171.15 Compound 3 

213.24 Compound 4  (or JAM317)

445.41 Compound 6 

391.38 Compound 7 

564.53 Compound 9 

326.39 Compound 11

439.55 Compound 13

369.18   Compound 14a

385.41  Compound 14b (or 5c)

Compound 15

371.39 Compound 17

375.33 Compound 20

199.21 Compound 22

270.28 Compound 23

343.42 Compound 25

312.36 Compound 26

385.50 Compound 28

425.52 Compound 29

329.31 Compound 30

343.33 Compound 31

357.37 Compound 32

371.39 Compound 34

385.42 Compound 35

327.25 Compound 36

355.30 Compound 38

399.45 Compound 40

413.47 Compound 42

371.39 Compound 44

 2.44 Compound 47

447.49   Compound 47a

 Compound 47b

Compound 49

357.36 Compound 51

618.69 Compound 52

660.73 Compound 56

469.54 Compound 57

511.58 Compound 59

511.48 Compound 60

464.19 A

618.54 B

602.54 C

530.47 D

334.38 E

484.51 F

525.51 G

509.51 LTP 073

255.23 JAM0351

693.66 JAM0359

679.63

Those skilled in the art will appreciate that the representativestructures of the DON prodrugs shown in Table 1 combined with theguidance disclosed herein will enable those skilled in the art tosynthesize prodrugs of other glutamine analogs, such as L-DONV,aza-serine, as are exemplified in the generic structures of formula (I).In other words, it should be understood that the prodrugs of otherglutamine antagonists, such as L-DONV, aza-serine, can be synthesizedwith the same substituents R₁, R₂ and R₂′ as the DON prodrugs shown inTable 1.

Accordingly, in one aspect the presently disclosed subject matterprovides a prodrug of a glutamine antagonist, or a pharmaceuticallyacceptable salt or ester thereof, the prodrug having a structure offormula (I):

wherein: X is selected from the group consisting of a bond, —O—, and—(CH₂)_(n)—, wherein n is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, and 8; R₁ is selected from the group consistingof H and a first prodrug-forming moiety capable of forming a salt or anester; and R₂ is H or a second prodrug-forming moiety capable of formingan amide linkage, a carbamate linkage, a phosphoramidate linkage or aphosphorodiamidate linkage with the nitrogen adjacent to R₂; R₂′ isselected from the group consisting of H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, or R₂ and R₂′ together form a ring structure comprising—C(═O)-G-C(═O)—, wherein G is selected from the group consisting ofC₁-C₈ alkylene, C₁-C₈ heteroalkylene, C₅-C₈ cycloalkylene, C₆-C₁₂arylene, C₅-C₁₄ heteroarylene, bivalent C₄-C₁₀ heterocycle, each ofwhich can be optionally substituted; or R₁ and R₂′ together form a 4- to6-membered heterocylic ring comprising the oxygen atom adjacent to R₁and the nitrogen atom adjacent to R₂′; provided that the compound has atleast one prodrug-forming moiety selected from the group consisting ofthe first and the second prodrug-forming moieties.

As used herein, the term “amide linkage” comprises a structurerepresented by the formula:

wherein R_(v) is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, andsubstituted heteroaryl.

As used herein, the term “carbamate linkage” comprises a structurerepresented by the formula:

wherein R_(w) is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, andsubstituted heteroaryl.

As used herein, the term “phosphoramidate linkage” comprises a structurerepresented by the formula:

wherein R_(x) and R_(x)′ are each independently selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkylamine, substitutedalkylamine, heteroaryl, and substituted heteroaryl.

As used herein, the term “phosphorodiamidate linkage” comprises astructure represented by the formula:

wherein R_(y) and R_(z) are each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substitutedalkenyl, cycloalkenyl, substituted cycloalkenyl, —(CR₃R₄)_(m)—Z,—(CR₃R₄)_(m)-Q-Z, aryl, substituted aryl, alkylamine, substitutedalkylamine, heteroaryl, substituted heteroaryl, and

In some embodiments, X is —CH₂—, and n is 1.

In other embodiments, X is —O—. In some embodiments, the prodrugcompound has both the first prodrug-forming moiety and the secondprodrug-forming moiety. In some embodiments, the glutamine analog is aglutamine antagonist, i.e., the prodrug is a prodrug of a glutamineanalog that antagonizes a glutamine pathway. Exemplary glutamineantagonists include, without limitation, 6-diazo-5-oxo-norleucine (DON),and aza-serine, and 5-diazo-4-oxo-L-norvaline (L-DONV).

In some embodiments, the presently disclosed subject matter provides aprodrug of DON. In some embodiments, the prodrug of DON has a structureof formula (I). In some embodiments, the presently disclosed subjectmatter provides a prodrug of L-DONV. In some embodiments, the prodrug ofL-DONV has a structure of formula (I). In some embodiments, thepresently disclosed subject matter provides a prodrug of azaserine. Insome embodiments, the prodrug of azaserine has a structure of formula(I).

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with a basic moiety and theterminal hydroxyl group forms a salt.

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with an alkyl group and theoxygen of an adjoining hydroxyl group forms an ester.

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with an alkyl group and thenitrogen adjoining the R₂′ group, forms an azlactone or an oxazolidone.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkenyl, substituted cycloalkenyl, tri(hydrocarbyl)ammonium, andtetra(hydrocarbyl)ammonium. Preferred alkyl group, cycloalkyl group,alkenyl group, alkynyl group, and cycloalkenyl group substituentsinclude alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.

In some embodiments, R₁ of formula (I) is not H. In some embodiments, R₁of formula (I) is not H when R₂ and R₂′ are H. In some embodiments, R₂and R₂′ of formula (I) are each H when and R₁ is not H.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of a C₁₋₆ straight-chain alkyl, a substituted C₁₋₆straight-chain alkyl, a C₁₋₆ branched alkyl, a substituted C₁₋₆ branchedalkyl, tri(C₁-C₈-alkyl)ammonium, tetra(C₁-C₈-alkyl)ammonium,triphenylammonium, tri(hydroxy-C₁-C₈-alkyl)ammonium, andtetra(hydroxy-C₁-C₈-alkyl)ammonium.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl,trimethylammonium, triethylammonium, tri(hydroxyethyl)ammonium,tripropylammonium, and tri(hydroxypropyl)ammonium. In some embodiments,R₁ of formula (I) is methyl. In some embodiments, R₁ of formula (I) isethyl. In some embodiments, R₁ of formula (I) is isopropyl.

In some embodiments, R₂ of formula (I) comprises a residue PRO₂ of thesecond prodrug-forming moiety, which, together with a carbonyl, oxycarbonyl, or phosphonyl group and the nitrogen of the adjoining NH,forms an amide, a carbamate, phosphoramidate, or phosphorodiamidatelinkage.

In some embodiments, R₂ of formula (I) comprises a moiety selected fromthe group consisting of an amino acid, an N-substituted amino acid, apeptide, a substituted peptide, a monocyclic ring, a substitutedmonocyclic ring, a bicyclic ring, a substituted bicyclic ring, a purinenucleoside, a substituted purine nucleoside, a pyrimidine nucleoside,and a substituted pyrimidine nucleoside.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein:

X is selected from the group consisting of a bond, —O—, and —(CH₂)_(n)—,wherein n is an integer selected from the group consisting of 1, 2, 3,4, 5, 6, 7, and 8;

R₁ is selected from the group consisting of H, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl; R₂ is an amino acid, an N-substituted aminoacid, or —C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀;

R₂′ is selected from the group consisting of H, C₁-C₆ alkyl, andsubstituted C₁-C₆ alkyl;

each R₃ and R₄ are independently H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, aryl, substituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8;

R₅ and R₆ are independently H or alkyl; and

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, monosaccharide, acylatedmonosaccharide, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein X is —CH₂—.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, having formula (I),wherein X is —O—.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein R₁ isselected from the group consisting of methyl, ethyl, isopropyl,cyclopentyl, cyclohexyl, trimethylammonium, triethylammonium,tri(hydroxyethyl)ammonium, tripropylammonium, andtri(hydroxypropyl)ammonium.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein:

R₂ is selected from the group consisting of —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆,and —C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀;

wherein:

Y is —O— or a bond;

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8; and

each R₃ and R₄ is independently H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl,aryl, or substituted aryl;

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein Y is a bond;m is 1; R₅ and R₆ are each H.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein R₂ is anamino acid.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein the aminoacid is tryptophan.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein R₂ is aN-acyl amino acid.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, wherein the aminoacid is tryptophan.

In another embodiment, the disclosure provides a compound having formula(IIA):

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, benzyl, p-hydroxybenzyl —CH₂CH(CH₃)₂, —CH₂OH, —CH(OH)CH₃,—CH₂-3-indoyl, —CH₂COOH, —CH₂CH₂COOH, —CH₂C(O)NH₂, —CH₂CH₂C(O)NH₂,—CH₂SH, —CH₂CH₂SCH₃, —(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂, and—CH₂-3-imidazoyl;

R₁₂ is selected from the group consisting of H, C₁₄ alkyl, and—C(═O)R₁₃; and

R₁₃ is C₁₄ alkyl.

In another embodiment, the disclosure provides a compound having formula(IIB):

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, benzyl, p-hydroxybenzyl —CH₂CH(CH₃)₂, —CH₂OH, —CH(OH)CH₃,—CH₂-3-indoyl, —CH₂COOH, —CH₂CH₂COOH, —CH₂C(O)NH₂, —CH₂CH₂C(O)NH₂,—CH₂SH, —CH₂CH₂SCH₃, —(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂, and—CH₂-3-imidazoyl;

R₁₂ is selected from the group consisting of H, C₁₋₄ alkyl, and—C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

In another embodiment, the disclosure provides a compound having formula(IIA) or formula (IIB), or a pharmaceutically acceptable salt thereof,wherein R₁ is selected from the group consisting of methyl, ethyl, andisopropyl.

In another embodiment, the disclosure provides a compound having formula(IIA) or formula (IIB), or a pharmaceutically acceptable salt thereof,wherein R₁₁ is selected from the group consisting of methyl, sec-butyl,benzyl, —CH₂CH(CH₃)₂, —CH(OH)CH₃ and —CH₂-3-indoyl. In anotherembodiment, R₁₁ is —CH₂-3-indoyl, i.e.,

In another embodiment, the disclosure provides a compound having formula(IIA) or formula (IIB), or a pharmaceutically acceptable salt thereof,wherein R₁₂ is selected from the group consisting of H and —C(═O)R₁₃. Inanother embodiment, R₁₂ is —C(═O)R₁₃ and R¹³ is methyl.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, selected from thegroup consisting of:

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, which is:

In another embodiment, the disclosure provides a compound having formula(III):

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₃ and R₄ are independently selected from the group consisting of H,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, aryl, and substituted aryl; and

R₁₀ is C₁₋₆ alkyl.

In another embodiment, the disclosure provides a compound having formula(III), or a pharmaceutically acceptable salt thereof, wherein R₁ isselected from the group consisting of methyl, ethyl, and isopropyl.

In another embodiment, the disclosure provides a compound having formula(III), or a pharmaceutically acceptable salt thereof, wherein R₃ isselected from the group consisting of methyl and phenyl; and R₄ is H.

In another embodiment, the disclosure provides a compound having formula(III), or a pharmaceutically acceptable salt thereof, wherein R₃ is Hand R₄ is selected from the group consisting of methyl and phenyl.

In another embodiment, the disclosure provides a compound having formula(III), or a pharmaceutically acceptable salt thereof, wherein R₁₀ isselected from the group consisting of isopropyl and tert-butyl.

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, selected from thegroup consisting of:

In another embodiment, the disclosure provides a compound having formula(I), or a pharmaceutically acceptable salt thereof, selected from thegroup consisting of:

In another embodiment, the disclosure provides a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of:

In another embodiment, the disclosure provides a pharmaceuticalcomposition comprising the compound of any one of formula (I), formula(IIA), formula (IIB), or formula (III), or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier, diluent, orexcipient.

In another embodiment, the disclosure provides a method of treatingcancer in a subject, the method comprising administering to the subjectin need thereof a compound of any one of formula (I), formula (IIA),formula (IIB), or formula (III), or a pharmaceutically acceptable saltthereof. In another embodiment, compound of any one of formula (I),formula (IIA), formula (IIB), or formula (III), or a pharmaceuticallyacceptable salt thereof, is administered subcutaneously to the subject.In another embodiment, the compound of formula (I), formula (IIA),formula (IIB), or formula (III) is administered in combination with oneor more additional anticancer agents. The term “anticancer agent” refersto radiotherapy, e.g., gamma-radiation, neutron beam radiotherapy,and/or any chemotherapeutic drug. In another embodiment, the compound offormula (I), formula (IIA), formula (IIB), or formula (III) isadministered as the only anticancer agent to the subject, i.e., thecompound of formula (I), formula (IIA), formula (IIB), or formula (III)is administered to the subject as a single anticancer agent. In anotherembodiment, R₁ is C₁₋₆ alkyl. In another embodiment, the cancer isselected from the group consisting of non-small cell lung cancer,melanoma, squamous cell carcinoma, kidney cancer, liver cancer,pancreatic cancer, colon cancer, triple negative breast cancer, andglioma. In another embodiment, the cancer is glioblastoma.

As used herein, the term “amino acid” includes moieties having acarboxylic acid group and an amino group. The term amino acid thusincludes both natural amino acids (including proteinogenic amino acids)and non-natural amino acids. The term “natural amino acid” also includesother amino acids that can be incorporated into proteins duringtranslation (including pyrrolysine and selenocysteine). Additionally,the term “natural amino acid” also includes other amino acids, which areformed during intermediary metabolism, e.g., ornithine generated fromarginine in the urea cycle. The natural amino acids are summarized inTable 2:

TABLE 2 Natural Amino Acids (Used For Protein Biosynthesis) Amino acid 3letter code 1-letter code Alanine ALA A Cysteine CYS C Aspartic Acid ASPD Glutamic Acid GLU E Phenylalanine PHE F Glycine GLY G Histidine HIS HIsoleucine ILE I Lysine LYS K Leucine LEU L Methionine MET M AsparagineASN N Proline PRO P Glutamine GLN Q Arginine ARG R Serine SER SThreonine THR T Valine VAL V Tryptophan TRP W Tyrosine TYR Y

The natural or non-natural amino acid may be optionally substituted. Inone embodiment, the amino acid is selected from proteinogenic aminoacids. Proteinogenic amino acids include glycine, alanine, valine,leucine, isoleucine, aspartic acid, glutamic acid, serine, threonine,glutamine, asparagine, arginine, lysine, proline, phenylalanine,tyrosine, tryptophan, cysteine, methionine and histidine. The term aminoacid includes alpha amino acids and beta amino acids, such as, but notlimited to, beta alanine and 2-methyl beta alanine. The term amino acidalso includes certain lactam analogues of natural amino acids, such as,but not limited to, pyroglutamine. The term amino acid also includesamino acids homologues including homocitrulline, homoarginine,homoserine, homotyrosine, homoproline and homophenylalanine.

The terminal portion of the amino acid residue or peptide may be in theform of the free acid i.e., terminating in a —COOH group or may be in amasked (protected) form, such as in the form of a carboxylate ester orcarboxamide. In certain embodiments, the amino acid or peptide residueterminates with an amino group. In an embodiment, the residue terminateswith a carboxylic acid group —COOH or an amino group —NH₂. In anotherembodiment, the residue terminates with a carboxamide group. In yetanother embodiment, the residue terminates with a carboxylate ester.

As disclosed hereinabove, the term “amino acid” includes compoundshaving a —COOH group and an —NH₂ group. A substituted amino acidincludes an amino acid which has an amino group which is mono- ordi-substituted. In particular embodiments, the amino group may bemono-substituted. (A proteinogenic amino acid may be substituted atanother site from its amino group to form an amino acid which is asubstituted proteinogenic amino acid). The term substituted amino acidthus includes N-substituted metabolites of the natural amino acidsincluding, but not limited to, N-acetyl cysteine, N-acetyl serine, andN-acetyl threonine.

For example, the term “N-substituted amino acid” includes N-alkyl aminoacids (e.g., C₁₋₆ N-alkyl amino acids, such as sarcosine,N-methyl-alanine, N-methyl-glutamic acid and N-tert-butylglycine), whichcan include C₁₋₆ N-substituted alkyl amino acids (e.g., N-(carboxyalkyl) amino acids (e.g., N-(carboxymethyl)amino acids) andN-methylcycloalkyl amino acids (e.g., N-methylcyclopropyl amino acids));N,N-di-alkyl amino acids (e.g., N,N-di-C₁₋₆ alkyl amino acids (e.g.,N,N-dimethyl amino acid)); N,N,N-tri-alkyl amino acids (e.g.,N,N,N-tri-C₁₋₆ alkyl amino acids (e.g., N,N,N-trimethyl amino acid));N-acyl amino acids (e.g., C₁₋₆N-acyl amino acid); N-aryl amino acids(e.g., N-phenyl amino acids, such as N-phenylglycine); N-amidinyl aminoacids (e.g., an N-amidine amino acid, i.e., an amino acid in which anamine group is replaced by a guanidino group).

The term “amino acid” also includes amino acid alkyl esters (e.g., aminoacid C₁₋₆ alkyl esters); and amino acid aryl esters (e.g., amino acidphenyl esters).

For amino acids having a hydroxy group present on the side chain, theterm “amino acid” also includes O-alkyl amino acids (e.g., C₁₋₆ O-alkylamino acid ethers); O-aryl amino acids (e.g., O-phenyl amino acidethers); O-acyl amino acid esters; and O-carbamoyl amino acids.

For amino acids having a thiol group present on the side chain, the term“amino acid” also includes S-alkyl amino acids (e.g., C₁₋₆ S-alkyl aminoacids, such as S-methyl methionine, which can include C₁₋₆ S-substitutedalkyl amino acids and S-methylcycloalkyl amino acids (e.g.,S-methylcyclopropyl amino acids)); S-acyl amino acids (e.g., a C₁₋₆S-acyl amino acid); S-aryl amino acid (e.g., a S-phenyl amino acid); asulfoxide analogue of a sulfur-containing amino acid (e.g., methioninesulfoxide) or a sulfoxide analogue of an S-alkyl amino acid (e.g.,S-methyl cystein sulfoxide) or an S-aryl amino acid.

In other words, the presently disclosed subject matter also envisagesderivatives of natural amino acids, such as those mentioned above whichhave been functionalized by simple synthetic transformations known inthe art (e.g., as described in “Protective Groups in Organic Synthesis”by T W Greene and P G M Wuts, John Wiley & Sons Inc. (1999)), andreferences therein.

Examples of non-proteinogenic amino acids include, but are not limitedto: citrulline, hydroxyproline, 4-hydroxyproline, β-hydroxyvaline,ornithine, β-amino alanine, albizziin, 4-amino-phenylalanine,biphenylalanine, 4-nitro-phenylalanine, 4-fluoro-phenylalanine,2,3,4,5,6-pentafluoro-phenylalanine, norleucine, cyclohexylalanine,α-aminoisobutyric acid, α-aminobutyric acid, α-aminoisobutyric acid,2-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid,selenomethionine, lanthionine, dehydroalanine, γ-amino butyric acid,naphthylalanine, aminohexanoic acid, pipecolic acid,2,3-diaminoproprionic acid, tetrahydroisoquinoline-3-carboxylic acid,tert-leucine, tert-butylalanine, cyclopropylglycine, cyclohexylglycine,4-aminopiperidine-4-carboxylic acid, diethylglycine, dipropylglycine andderivatives thereof wherein the amine nitrogen has been mono- ordi-alkylated.

The term “peptide” refers to an amino acid chain consisting of 2 to 9amino acids, unless otherwise specified. In preferred embodiments, thepeptide used in the present invention is 2 or 3 amino acids in length.In one embodiment, a peptide can be a branched peptide. In thisembodiment, at least one amino acid side chain in the peptide is boundto another amino acid (either through one of the termini or the sidechain).

The term “N-substituted peptide” refers to an amino acid chainconsisting of 2 to 9 amino acids in which one or more NH groups aresubstituted, e.g., by a substituent described elsewhere herein inrelation to substituted amino groups. Optionally, the N-substitutedpeptide has its N-terminal amino group substituted and, in oneembodiment, the amide linkages are unsubstituted.

In one embodiment, an amino acid side chain is bound to another aminoacid. In a further embodiment, side chain is bound to the amino acid viathe amino acid's N-terminus, C-terminus, or side chain.

Examples of natural amino acid sidechains include hydrogen (glycine),methyl (alanine), isopropyl (valine), sec-butyl (isoleucine),—CH₂CH(CH₃)₂ (leucine), benzyl (phenylalanine), p-hydroxybenzyl(tyrosine), —CH₂OH (serine), —CH(OH)CH₃ (threonine), —CH₂-3-indoyl(tryptophan), —CH₂COOH (aspartic acid), —CH₂CH₂COOH (glutamic acid),—CH₂C(O)NH₂ (asparagine), —CH₂CH₂C(O)NH₂ (glutamine), —CH₂SH,(cysteine), —CH₂CH₂SCH₃ (methionine), —(CH₂)₄NH₂ (lysine),—(CH₂)₃NHC(═NH)NH₂(arginine) and —CH₂-3-imidazoyl (histidine).

Exemplary monocyclic rings and bicyclic rings include, withoutlimitation, benzene, pyrimidines, and purines, and more generally aryland heteroaryl rings. Exemplary heteroaryls include, but are not limitedto, pyridyl, pyrimidinyl, pyrazinyl, furanyl, thienyl, pyrazolyl,oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, pyrrolyl, imidazolyl,indolyl, indolinolyl, and imidazopyridazinyl. Aryls include phenyl (C₆),benzyl, naphthyl (C₁₀), and biphenyl (C₁₂). Exemplary pyrimidinesinclude, without limitation, cytosine, thymine, and uracil. Exemplarypurines include, without limitation, purine, adenine, N-substitutedadenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uricacid, and isoguanine. Exemplary purine nucleosides include, withoutlimitation, adenine and guanine.

In some embodiments, R₂ of formula (I) is selected from the groupconsisting of H, alkyl, —C(═O)—Ar, —C(═O)—Y—(CR₃R₄)_(m)—Ar,—C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆, —P(═O)(OR₇)_(n)(NHR₉)_(o),—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—R₈, —C(═O)—Y—(CR₃R₄)_(m)—Ar—O—R₈,—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀, —C(═O)—O—R₉,—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—Ar, and —C(═O)—Y—(CR₃R₄)_(m)—Ar—NR₅R₆;wherein: Y is —O— or a bond; m is an integer selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; each n and o is an integerfrom 0 to 2 provided that the sum of n and o is 2; R₃ and R₄ isindependently H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl, aryl orsubstituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

each R₅ and R₆ is independently H, alkyl, —C(═O)—(CR₃R₄)_(m)H,—C(═O)—(NR₅R₆), or —C(═O)—(CR₃R₄)_(m)—NR₅R₆; each R₇ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, —(CR₃R₄)_(m)—Z, —(CR₃R₄)_(m)-Q-Z, wherein Q is amonosaccharide, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, and wherein Z is

or wherein R₇ together with the oxygen atom to which it is attachedforms a purine or pyrimidine nucleoside; each R₉ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, —(CR₃R₄)_(m)—Z, aryl, substituted aryl, heteroaryl,substituted heteroaryl, and

wherein R₁ and X are as defined above, provided that R₁ is not H;

each R₈ is independently alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl,substituted aryl, heteroaryl, substituted heteroaryl; each R₁₀ isindependently alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, monosaccharide, acylated monosaccharide, aryl, substitutedaryl, heteroaryl, substituted heteroaryl; and Ar is aryl, substitutedaryl, heteroaryl, or substituted heteroaryl. It should be appreciatedthat in addition to substitutions on the amino group of Z, one or moresubstitutions R₃, R₄, R₅, and/or R₆ can be made to the 5 or 6 memberedrings of Z.

In particular embodiments Ar is

In other particular embodiments, Ar is and

In yet other particular embodiments. Ar is benzyl.

In particular embodiments, the prodrug compound of formula (I) is

In some embodiments, R₂ of formula (I) is —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆;wherein: (i) Y is a bond; m is 1; R₅ and R₆ are each H; (ii) Y is abond; m is 1; R₅ is H; R₆ is —C(═O)—(CR₃R₄)_(m)H.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In such embodiments, it should be appreciated that the terminal carboxygroups of the compounds of formula (I) shown above can be used to formsalts. In an exemplary embodiment, a salt of any of the compounds offormula (I) shown above can be formed when an H is replaced by NEt₃, aswill be appreciated by those skilled in the art.

In some embodiments, R₂ of formula (I) is —P(═O)(OR₇)_(n)(NHR₉)_(o);wherein: n is 2 and o is 0; n is 1 and o is 1; or n is 0 and o is 2.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—R₈ or —C(═O)—Y—(CR₃R₄)_(m)—Ar—O—R₈,wherein: Y is —O—; m is 0; and Ar is benzyl.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀; wherein: (i) m is 1; R₃ is H; and R₄is methyl, iPr, or aryl; (ii) m is 1; R₃ and R₄ are each H; or (vi) m is1; R₃ and R₄ are each methyl.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In particular embodiments, the prodrug compound of formula (I) is offormula:

wherein R₁ is as defined above.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆;wherein Y is a bond; each m is 1; each R₅ is H; each R₆ is independentlyH or —C(═O)—(CR₃R₄)_(m)—NR₅R₆.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is —C(═O)—Ar, or—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—Ar.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is —C(═O)—Y—(CR₃R₄)_(m)—Ar—NR₅R₆;wherein: Y is 0; each m is independently 0, 1, or 3; each R₃ isindependently H, C₁-C₆ alkyl, or —(CR₃R₄)_(m)—NR₅R₆; each R₄ is H; eachR₅ is independently H, —C(═O)—(CR₃R₄)_(m)H, —C(═O)—NR₅R₆, or—C(═O)—(CR₃R₄)_(m)—NR₅R₆; each R₆ is H.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆,wherein: Y is a bond; m is 1, 2 or 3; each R₃ is independently H, C₁-C₆alkyl, or —(CR₃R₄)_(m)—NR₅R₆; each R₄ is H; each R₅ is independently H,—C(═O)—(CR₃R₄)_(m)H, —C(═O)—(NR₅R₆), or —C(═O)—(CR₃R₄)_(m)—NR₅R₆; eachR₆ is H.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

In some embodiments, R₂ of formula (I) is H and R₁ is selected from thegroup consisting of alkyl and substituted alkyl.

In particular embodiments, the prodrug compound of formula (I) isselected from the group consisting of:

It should be appreciated that all of the compounds of formula (I)depicted in the structural formulas as prodrugs of DON are merelyexemplary of prodrugs of glutamine analogs, such as L-DONV andaza-serine, that can be synthesized using the guidance of the presentlydisclosed subject matter.

The disclosure also provides the following embodiments numberedEmbodiments I-XXXIII.

Embodiment I

A prodrug of a glutamine analog, or a pharmaceutically acceptable saltor ester thereof, the prodrug having a structure of formula (I):

wherein:

X is selected from the group consisting of a bond, —O—, and —(CH₂)_(n)—,wherein n is an integer selected from the group consisting of 1, 2, 3,4, 5, 6, 7, and 8;

R₁ is selected from the group consisting of H and a firstprodrug-forming moiety capable of forming a salt or an ester; and

R₂ is H or a second prodrug-forming moiety capable of forming an amidelinkage, a carbamate linkage, a phosphoramidate linkage or aphosphorodiamidate linkage with the nitrogen adjacent to R₂;

R₂′ is selected from the group consisting of H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, or

R₂ and R₂′ together form a ring structure comprising —C(═O)-G-C(═O)—,wherein G is selected from the group consisting of C₁-C₈ alkylene, C₁-C₈heteroalkylene, C₅-C₈ cycloalkylene, C₆-C₁₂ arylene, C₅-C₁₄heteroarylene, bivalent C₄-C₁₀ heterocycle, each of which can beoptionally substituted; or

R₁ and R₂′ together form a 4- to 6-membered heterocylic ring comprisingthe oxygen atom adjacent to R₁ and the nitrogen atom adjacent to R₂′;

provided that the compound has at least one prodrug-forming moietyselected from the group consisting of the first and the secondprodrug-forming moieties.

Embodiment II

The prodrug of Embodiment I, wherein X is —CH₂—.

Embodiment III

The prodrug of Embodiment I, wherein X is —O—.

Embodiment IV

The prodrug of Embodiment I, wherein the glutamine analog is a glutamineantagonist selected from the group consisting of6-diazo-5-oxo-norleucine (DON), 5-diazo-4-oxo-L-norvaline (L-DONV), andaza-serine.

Embodiment V

The prodrug of Embodiment I, wherein R₁ comprises a residue PRO₁ of theprodrug-forming moiety, which, together with:

(i) a basic moiety and a terminal hydroxyl group forms a salt;

(ii) an alkyl group and the oxygen of an adjoining hydroxyl group formsan ester; or

(iii) an alkyl group and the nitrogen atom adjoining R₂′ forms anazlactone or an oxazolidone.

Embodiment VI

The prodrug of Embodiment I, wherein R₁ is selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkenyl, substituted cycloalkenyl, tri(hydrocarbyl)ammonium, andtetra(hydrocarbyl)ammonium.

Embodiment VII

The prodrug of Embodiment VI, wherein R₁ is selected from the groupconsisting of a C₁₋₆ straight-chain alkyl, a substituted C₁₋₆straight-chain alkyl, a C₁₋₆ branched alkyl, a substituted C₁₋₆ branchedalkyl, tri(C₁-C₈-alkyl)ammonium, tetra(C₁-C₈-alkyl)ammonium,triphenylammonium, tri(hydroxy-C₁-C₈-alkyl)ammonium, andtetra(hydroxy-C₁-C₈-alkyl)ammonium.

Embodiment VIII

The prodrug of Embodiment I, wherein R₁ is selected from the groupconsisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl,trimethylammonium, triethylammonium, tri(hydroxyethyl)ammonium,tripropylammonium, and tri(hydroxypropyl)ammonium.

Embodiment IX

The prodrug of Embodiment I, wherein R₂ comprises a residue PRO₂ of thesecond prodrug-forming moiety which comprises a carbonyl, an oxycarbonyl, or a phosphonyl group, wherein the carbonyl, the oxy carbonyl,or the phosphonyl group is bound to the nitrogen of the adjoining NR₂′to form an amide, a carbamate, phosphoramidate, or phosphorodiamidatelinkage.

Embodiment X

The prodrug of Embodiment IX, wherein PRO₂ comprises a moiety selectedfrom the group consisting of an amino acid, an N-substituted amino acid,a peptide, a substituted peptide, a monocyclic ring, a substitutedmonocyclic ring, a bicyclic ring, a substituted bicyclic ring, a purinenucleoside, a substituted purine nucleoside, a pyrimidine nucleoside,and a substituted pyrimidine nucleoside.

Embodiment XI

The prodrug of Embodiment I, wherein R₂ is selected from the groupconsisting of H, alkyl, —C(═O)—Ar, —C(═O)—Y—(CR₃R₄)_(m)—Ar,—C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆, —P(═O)(OR₇)_(n)(NHR₉)_(o),—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—R₈, —C(═O)—Y—(CR₃R₄)_(m)—Ar—O—R₈,—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀, —C(═O)—O—R₉,—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—Ar, and —C(═O)—Y—(CR₃R₄)_(m)—Ar—NR₅R₆;

wherein:

Y is —O— or a bond;

m is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8;

each n and o is an integer from 0 to 2 provided that the sum of n and ois 2;

each R₃ and R₄ is independently H, C₁-C₆ alkyl or substituted C₁-C₆alkyl, aryl or substituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

each R₅ and R₆ is independently H, alkyl, —C(═O)—(CR₃R₄)_(m),—C(═O)—(NR₅R₆), or —C(═O)—(CR₃R₄)_(m)—NR₅R₆;

each R₇ is independently selected from the group consisting of H, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, —(CR₃R₄)_(m)—Z, —(CR₃R₄)_(m)-Q-Z wherein Q isa monosaccharide, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, and wherein Z is

or wherein R₇ together with the oxygen atom to which it is attachedforms a purine or pyrimidine nucleoside;

each R₉ is independently selected from the group consisting of H, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, —(CR₃R₄)_(m)—Z, aryl, substituted aryl,heteroaryl, substituted heteroaryl, and

wherein R₁ and X are as defined in Embodiment 1, provided that R₁ is notH,

each R₈ is independently alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl,substituted aryl, heteroaryl, substituted heteroaryl;

each R₁₀ is independently alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl,substituted aryl, heteroaryl, substituted heteroaryl; and

Ar is aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

Embodiment XII

The prodrug of Embodiment XI, wherein the compound of formula (I) is

Embodiment XIII

The prodrug of Embodiment XI, wherein R₂ is —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆;

wherein:

(i) Y is a bond;

m is 1;

R₅ and R₆ are each H; or

(ii) Y is a bond;

m is 1;

R₅ is H;

R₆ is —C(═O)—(CR₃R₄)_(m).

Embodiment XIV

The prodrug of Embodiment XIII, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XV

The prodrug of Embodiment XI, wherein R₂ is —P(═O)(OR₇)_(n)(NHR₉)_(o);

wherein:

(i) n is 2 and o is 0;

(ii) n is 1 and o is 1; or

(iii) n is 0 and o is 2.

Embodiment XVI

The prodrug of Embodiment XV, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XVII

The prodrug of Embodiment XI, wherein: R₂ is—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—R₈ or —C(═O)—Y—(CR₃R₄)_(m)—Ar—O—R₈; and

Y is —O—;

m is 0; and

Ar is benzyl.

Embodiment XVIII

The prodrug of Embodiment XVII, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XIX

The prodrug of Embodiment XI, wherein R₂ is—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀;

wherein:

(i) m is 1;

R₃ is H; and

R₄ is methyl, iPr, or aryl;

(ii) m is 1;

R₃ and R₄ are each H; or

(iii) m is 1;

R₃ and R₄ are each methyl.

Embodiment XX

The prodrug of Embodiment XIX, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XXI

The prodrug of Embodiment XI, wherein the compound of formula (I) is offormula:

wherein R₁ is as defined above in Embodiment I.

Embodiment XXII

The prodrug of Embodiment XXI, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XXIII

The prodrug of Embodiment XI, wherein R₂ is —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆;wherein

Y is a bond;

each m is 1;

each R₅ is H;

each R₆ is independently H or —C(═O)—(CR₃R₄)_(m)—NR₅R₆.

Embodiment XXIV

The prodrug of Embodiment XXIII, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XXV

The prodrug of Embodiment XI, wherein R₂ is —C(═O)—Ar, or—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—Ar.

Embodiment XXVI

The prodrug of Embodiment XXV, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XXVII

The prodrug of Embodiment XI, wherein R₂ is—C(═O)—Y—(CR₃R₄)_(m)—Ar—NR₅R₆; wherein:

Y is O;

each m is independently 0, 1, or 3;

each R₃ is independently H, C₁-C₆ alkyl, or —(CR₃R₄)_(m)—NR₅R₆;

each R₄ is H;

each R₅ is independently H, —C(═O)—(CR₃R₄)_(m), —C(═O)—NR₅R₆, or—C(═O)—(CR₃R₄)_(m)—NR₅R₆;

each R₆ is H.

Embodiment XXVIII

The prodrug of Embodiment XXVII, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XXIX

The prodrug of Embodiment XI, wherein R₂ is —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆,wherein:

Y is a bond;

each m is independently 1, 2 or 3;

each R₃ is independently H, C₁-C₆ alkyl, or —(CR₃R₄)_(m)—NR₅R₆, each R₄is H;

each R₅ is independently H, —C(═O)—(CR₃R₄)_(m), —C(═O)—(NR₅R₆), or—C(═O)—(CR₃R₄)_(m)—NR₅R₆;

each R₆ is H.

Embodiment XXX

The prodrug of Embodiment XXIX, wherein the compound of formula (I) isselected from the group consisting of:

Embodiment XXXI

The prodrug of Embodiment XI, wherein R₂ is H and R₁ is selected fromthe group consisting of alkyl and substituted alkyl.

Embodiment XXXII

The prodrug of Embodiment XXXI, wherein the compound of formula (I) isselected from the group, consisting of:

Embodiment XXXIII

A pharmaceutical composition comprising a compound of formula (I), and apharmaceutically acceptable carrier, diluent, or excipient.

II. Pharmaceutical Compositions and Administration

In another aspect, the present disclosure provides a pharmaceuticalcomposition including one prodrug compound of formula (I), alone or incombination with one or more additional therapeutic agents in admixturewith a pharmaceutically acceptable excipient. Accordingly, in someembodiments, the presently disclosed subject matter provides apharmaceutical composition comprising a compound of formula (I), and apharmaceutically acceptable carrier, diluent, or excipient. One of skillin the art will recognize that the pharmaceutical compositions includethe pharmaceutically acceptable salts of the compounds described above.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and include salts of active compounds whichare prepared with relatively nontoxic acids or bases, depending on theparticular substituent moieties found on the compounds described herein.When compounds of the present disclosure contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent or by ion exchange,whereby one basic counterion (base) in an ionic complex is substitutedfor another. Examples of pharmaceutically acceptable base addition saltsinclude sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt.

When compounds of the present disclosure contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent or by ion exchange,whereby one acidic counterion (acid) in an ionic complex is substitutedfor another. Examples of pharmaceutically acceptable acid addition saltsinclude those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids, such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al, “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present disclosure contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

Accordingly, pharmaceutically acceptable salts suitable for use with thepresently disclosed subject matter include, by way of example but notlimitation, acetate, benzenesulfonate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000).

In particular embodiments, the salt is a tri(hydrocarbyl)ammonium ortetra(hydrocarbyl)ammonium salt. In yet more particular embodiments, thesalt is selected from the group consisting of atri(C₁-C₈-alkyl)ammonium, tetra(C₁-C₈-alkyl)ammonium, triphenylammonium,tri(hydroxy-C₁-C₈alkyl)ammonium, and tetra(hydroxy-C₁-C₈alkyl)ammoniumsalt. In even more particular embodiments, the salt is selected from thegroup consisting of a trimethylammonium, triethylammonium,tri(hydroxyethyl)ammonium, tripropylammonium, andtri(hydroxypropyl)ammonium salt.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including oral (sublingual, buccal), peroral, sublingual, systemic andtopical or localized administration. Techniques and formulationsgenerally may be found in Remington: The Science and Practice ofPharmacy (20^(th) ed.) Lippincott, Williams & Wilkins (2000).

Depending on the specific conditions being treated, such agents may beformulated into liquid (e.g., solutions, suspensions, or emulsions) orsolid dosage forms (capsules or tablets) and administered systemicallyor locally. The agents may be delivered, for example, in a timed-,controlled, or sustained-slow release form as is known to those skilledin the art. Techniques for formulation and administration may be foundin Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery. In some embodiments, thepharmaceutical composition is administered orally. In some embodiments,the pharmaceutical composition is administered intravenously. In someembodiments, the pharmaceutical composition is administeredintramuscularly. In some embodiments, the pharmaceutical composition isadministered intrathecally. In some embodiments, the pharmaceuticalcomposition is administered subcutaneously.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers, such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances, such as saline; preservatives, suchas benzyl alcohol; absorption promoters; and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Generally, the compounds according to the disclosure are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg perday, and from 5 to 40 mg per day are examples of dosages that may beused. A non-limiting dosage is 10 to 30 mg per day. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, the bioavailability of the compound(s), theadsorption, distribution, metabolism, and excretion (ADME) toxicity ofthe compound(s), and the preference and experience of the attendingphysician.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers, such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with filler,such as lactose, binders, such as starches, and/or lubricants such, astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

III. Methods for Treating a Disease or Disorder

The presently disclosed compounds are, which are orally bioavailable,less toxic prodrugs of glutamine analogs that are glutamine antagonists,allow a clinically acceptable dosing paradigm for diseases or conditionswherein excess and/or aberrant glutamine activity is implicated. As usedherein, the term “glutamine antagonist” refers to a glutamine analogthat interferes with a glutamine metabolic pathway, e.g., the inhibitionor blocking of a metabolic pathway downstream of glutamine in whichglutamine acts as a precursor of one or more non-glutamine compounds.Examples of such metabolic pathways are well known (see, e.g., Hensleyet al., “Glutamine and cancer: cell biology, physiology, and clinicalopportunities” J Clin Invest. 2013; 123(9):3678-3684; DeBerardinis etal., “Q's next: the diverse functions of glutamine in metabolism, cellbiology and cancer” Oncogene. 2009; 29(3):313-324; and Medina et al.,“Relevance of glutamine metabolism to tumor cell growth” Mol CellBiochem. 1992; 113(1):1-15). In some contexts, the term glutamineantagonist also includes glutamine analogs that inhibit glutamine uptakeby cells, thereby reducing its biological activity. Diseases orconditions wherein excess and/or aberrant glutamine activity isimplicated include, but are not limited to, infection, cancer,autoimmune diseases, and neurodegenerative or neurological diseases andother central nervous system disorders.

In general, the presently disclosed methods result in a decrease in theseverity of a disease or condition in a subject. The term “decrease” ismeant to inhibit, suppress, attenuate, diminish, arrest, or stabilize asymptom of a disease or condition.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disease or condition, and/orsymptoms associated therewith. It will be appreciated that, although notprecluded, treating a disease or condition does not require that thedisorder, condition or symptoms associated therewith be completelyeliminated.

Accordingly, in some embodiments, the presently disclosed subject matterprovide a method for treating a disease or a condition, the methodcomprising administering to a subject in need of treatment thereof, acompound of formula (I), or a pharmaceutical composition of any thereof,in an amount effective for treating the disease or condition.

The presently disclosed subject matter contemplates using a prodrug offormula (I), or a pharmaceutical composition comprising the prodrug offormula (I), optionally together with at least one antimicrobial agent(e.g., antibiotic, antiviral, and the like), to treat an infection.

As used herein, “infection” refers to the invasion of a host organism'sbodily tissues by disease-causing organisms, their multiplication, andthe reaction of host tissues to these organisms and the toxins theyproduce. Infectious disease, such as infection by any bacteria or virus,is contemplated for treatment using a compound of formula (I), or apharmaceutical composition of any thereof.

In some embodiments, the infection comprises a bacterial infection.Antibacterial effects of DON have been demonstrated in E. coli (seeCoggin et al., “6-Diazo-5-Oxo-L-Norleucine Inhibition of Escherichiacoli,” Journal of Bacteriology. 1965; 86). In some embodiments, thecompound of formula (I), or pharmaceutical composition of any thereof,inhibits bacterial growth and/or survival.

In some embodiments, the infection comprises a viral infection. Theantiviral effects of glutamine analogs, such as DON, have beendemonstrated (see, e.g., Cinatl et al., “Antiviral effects of6-diazo-5-oxo-L-norleucine on replication of herpes simplex virus type1” Antiviral Research. 1997; 33:165-175; Nishio et al., “Antiviraleffect of 6-diazo-5-oxo-L-norleucine, antagonist of γ-glutamyltranspeptidase, on replication of human parainfluenza virus type 2,”Journal of General Virology. 1990; 71:61-67). Examples of viralinfections contemplated for treatment using a compound of formula (I),or a pharmaceutical composition of any thereof include, withoutlimitation, herpes simplex virus type 1 (HSV-1), herpes simplex virustype 2 (HSV-2), human cytomegalovirus (HCMV), human parainfluenza virustype 2 (HPIV-2), Maloney leukemia virus (MLV), mumps, paramyxovirus,poliovirus, reovirus type 3, respiratory syncytial virus (RSV), Sendaivirus, and vesicular stomatitis virus (VSV).

In some embodiments, the compound of formula (I), or pharmaceuticalcomposition of any thereof, inhibits viral replication. In someembodiments, the compound of formula (I), or pharmaceutical compositionof any thereof, inhibits replication of herpes simplex virus type 1(HSV-1). In some embodiments, the compound of formula (I), orpharmaceutical composition of any thereof, inhibits replication ofherpes simplex virus type 2 (HSV-2). In some embodiments, the compoundof formula (I), or pharmaceutical composition of any thereof, inhibitsreplication of human cytomegalovirus (HCMV). In some embodiments, thecompound of formula (I), or pharmaceutical composition of any thereof,inhibits replication of human parainfluenza virus type 2 (HPIV-2). Insome embodiments, the compound of formula (I), or pharmaceuticalcomposition of any thereof, inhibits replication of Maloney leukemiavirus (MLV). In some embodiments, the compound of formula (I), orpharmaceutical composition of any thereof, inhibits replication ofmumps. In some embodiments, the compound of formula (I), orpharmaceutical composition of any thereof, inhibits replication ofparamyxovirus. In some embodiments, the compound of formula (I), orpharmaceutical composition of any thereof, inhibits replication ofpoliovirus. In some embodiments, the compound of formula (I), orpharmaceutical composition of any thereof, inhibits replication ofreovirus type 3. In some embodiments, the compound of formula (I), orpharmaceutical composition of any thereof, inhibits replication ofrespiratory syncytial virus (RSV). In some embodiments, the compound offormula (I), or pharmaceutical composition of any thereof, inhibitsreplication of Sendai virus. In some embodiments, the compound offormula (I), or pharmaceutical composition of any thereof, inhibitsreplication of vesicular stomatitis virus (VSV).

In some embodiments, the viral infection is influenza. As used herein,“influenza” refers to influenza A, B, or C, parainfluenza viruses, andany other influenza-like virus (see, e.g., U.S. Publication No.2006/0276438, incorporated by reference herein in its entirety, whichdiscloses using DON, and azaserine for treatment of influenza).

In an aspect, the presently disclosed subject matter involves the use ofa compound of formula (I), or a pharmaceutical composition thereof,optionally together with an antiviral agent, for the manufacture of amedicament for treating a viral infection and/or inhibiting replication.

As used herein, “antiviral agent” includes a compound that inhibits thereplication of viruses in cells, tissues, or organisms. Examples ofantiviral agents contemplated for use in combination with a prodrug offormula (I), or a pharmaceutical composition comprising a prodrug offormula (I) include, but are not limited to, Acyclovir(2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one),Valacyclovir (L-valine,2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy]ethyl ester,Pencyclovir (9-[4-hydroxy-3-(hydroxymethylbutyl)]guanine), Famcyclovir(2-[2-(amino-9H-purin-9-yl)]ethyl-1,3-propanediol diacetate), Ribavirin(1-beta-D-ribofuanosyl-1-H-1,2,4-triazol-3-carboxamide), Lamivudine((2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidine-2-one),Amantadine (1-amantanamine hydrochloride), and Rimantadine(α-methyltricyclo (3.3.1.1/3.7 decane-1-methylamine hydrochloride).

The presently disclosed subject matter contemplates using a prodrug offormula (I), or a pharmaceutical composition comprising the prodrug offormula (I), optionally together with at least one chemotherapeuticagent, at least one radiotherapeutic agent, and/or at least oneimmunotherapeutic agent to treat cancer. In some embodiments, suchtreatment includes treatment with any combination of radiotherapy,immunotherapy, photodynamic therapy, proton therapy, and/or surgery.

A “chemotherapeutic agent” is used to connote a compound or compositionthat is administered in the treatment of cancer. Chemotherapeutic agentscontemplated for use in combination with a prodrug of formula (I), or apharmaceutical composition comprising a prodrug of formula (I) include,but are not limited to, alkylating agents, such as thiotepa andcyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan andpiposulfan; aziridines, such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards, such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas, such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics, such as aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens, such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals,such as aminoglutethimide, mitotane, trilostane; folic acidreplenishers, such as folinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;edatraxate; defofamine; demecolcine; diaziquone; elformithine;elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g., paclitaxel and docetaxel;chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinumanalogs, such as cisplatin and carboplatin; vinblastine; platinum;etoposide; ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine; retinoic acid; esperamicins; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Chemotherapeutic agents also include anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide, and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese.

In some embodiments, the chemotherapeutic agent is an anti-metabolite.An anti-metabolite is a chemical with a structure that is similar to ametabolite required for normal biochemical reactions, yet differentenough to interfere with one or more normal functions of cells, such ascell division. Anti-metabolites include, but are not limited to,gemcitabine, fluorouracil, capecitabine, methotrexate sodium,ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine,5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine,pentostatin, fludarabine phosphate, and cladribine, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof.

As used herein, the term “immunotherapeutic agent” refers to a moleculethat can aid in the treatment of a disease by inducing, enhancing, orsuppressing an immune response in a cell, tissue, organ or subject.Examples of immunotherapeutic agents contemplated for use in combinationwith a prodrug of formula (I), or a pharmaceutical compositioncomprising a prodrug of formula (I) include, but are not limited to,immune checkpoint molecules (e.g., antibodies to immune checkpointproteins), interleukins (e.g., IL-2, IL-7, IL-12, IL-15), cytokines(e.g., interferons, G-CSF, imiquimod), chemokines (e.g., CCL3, CCL26,CXCL7), vaccines (e.g., peptide vaccines, dendritic cell (DC) vaccines,EGFRvIII vaccines, mesothilin vaccine, G-VAX, listeria vaccines), andadoptive T cell therapy including chimeric antigen receptor T cells (CART cells).

As used herein, “radiotherapeutic agent” means an agent which may beused in radiotherapy that acts through damaging cells (e.g., malignantcells) as a target through radiation irradiation. An exemplaryradiotherapeutic agent contemplated for use in combination with aprodrug of formula (I), or a pharmaceutical composition comprising aprodrug of formula (I) is the titanium peroxide contained in thesubstrate particle which generates a hydroxyl radial through radiationirradiation, and the hydroxyl radial exerts an action of attacking atarget, as described in U.S. Publication No. 2013/0017266, which isincorporated by reference herein in its entirety.

As used herein, a “cancer” in a patient refers to the presence of cellspossessing characteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells, for example, leukemic cells. A “tumor,” as usedherein, refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all precancerous and cancerous cells andtissues. A “solid tumor,” as used herein, is an abnormal mass of tissuethat generally does not contain cysts or liquid areas. A solid tumor maybe in the brain, colon, breasts, prostate, liver, kidneys, lungs,esophagus, head and neck, ovaries, cervix, stomach, colon, rectum,bladder, uterus, testes, and pancreas, as non-limiting examples. In someembodiments, the solid tumor regresses or its growth is slowed orarrested after the solid tumor is treated with the presently disclosedmethods. In other embodiments, the solid tumor is malignant. In someembodiments, the cancer comprises Stage 0 cancer. In some embodiments,the cancer comprises Stage I cancer. In some embodiments, the cancercomprises Stage II cancer. In some embodiments, the cancer comprisesStage III cancer. In some embodiments, the cancer comprises Stage IVcancer. In some embodiments, the cancer is refractory and/or metastatic.For example, the cancer may be refractory to treatment withradiotherapy, chemotherapy or monotreatment with immunotherapy. Canceras used herein includes newly diagnosed or recurrent cancers, includingwithout limitation, acute lymphoblastic leukemia, acute myelogenousleukemia, advanced soft tissue sarcoma, brain cancer, metastatic oraggressive breast cancer, breast carcinoma, bronchogenic carcinoma,choriocarcinoma, chronic myelocytic leukemia, colon carcinoma,colorectal carcinoma, Ewing's sarcoma, gastrointestinal tract carcinoma,glioma, glioblastoma multiforme, head and neck squamous cell carcinoma,hepatocellular carcinoma, Hodgkin's disease, intracranialependymoblastoma, large bowel cancer, leukemia, liver cancer, lungcarcinoma, Lewis lung carcinoma, lymphoma, malignant fibroushistiocytoma, a mammary tumor, melanoma, mesothelioma, neuroblastoma,osteosarcoma, ovarian cancer, pancreatic cancer, a pontine tumor,premenopausal breast cancer, prostate cancer, rhabdomyosarcoma,reticulum cell sarcoma, sarcoma, small cell lung cancer, a solid tumor,stomach cancer, testicular cancer, and uterine carcinoma.

In some embodiments, the cancer is acute leukemia. In some embodiments,the cancer is acute lymphoblastic leukemia. In some embodiments, thecancer is acute myelogenous leukemia. In some embodiments, the cancer isadvanced soft tissue sarcoma. In some embodiments, the cancer is a braincancer. In some embodiments, the cancer is breast cancer (e.g.,metastatic or aggressive breast cancer). In some embodiments, the canceris breast carcinoma. In some embodiments, the cancer is bronchogeniccarcinoma. In some embodiments, the cancer is choriocarcinoma. In someembodiments, the cancer is chronic myelocytic leukemia. In someembodiments, the cancer is a colon carcinoma (e.g., adenocarcinoma). Insome embodiments, the cancer is colorectal cancer (e.g., colorectalcarcinoma). In some embodiments, the cancer is Ewing's sarcoma. In someembodiments, the cancer is gastrointestinal tract carcinoma. In someembodiments, the cancer is a glioma. In some embodiments, the cancer isglioblastoma multifome. In some embodiments, the cancer is head and necksquamous cell carcinoma. In some embodiments, the cancer ishepatocellular carcinoma. In some embodiments, the cancer is Hodgkin'sdisease. In some embodiments, the cancer is intracranialependymoblastoma. In some embodiments, the cancer is large bowel cancer.In some embodiments, the cancer is leukemia. In some embodiments, thecancer is liver cancer. In some embodiments, the cancer is lung cancer(e.g., lung carcinoma). In some embodiments, the cancer is Lewis lungcarcinoma. In some embodiments, the cancer is lymphoma. In someembodiments, the cancer is malignant fibrous histiocytoma. In someembodiments, the cancer comprises a mammary tumor. In some embodiments,the cancer is melanoma. In some embodiments, the cancer is mesothelioma.In some embodiments, the cancer is neuroblastoma. In some embodiments,the cancer is osteosarcoma. In some embodiments, the cancer is ovariancancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer comprises a pontine tumor. In some embodiments,the cancer is premenopausal breast cancer. In some embodiments, thecancer is prostate cancer. In some embodiments, the cancer isrhabdomyosarcoma. In some embodiments, the cancer is reticulum cellsarcoma. In some embodiments, the cancer is sarcoma. In someembodiments, the cancer is small cell lung cancer. In some embodiments,the cancer comprises a solid tumor. In some embodiments, the cancer isstomach cancer. In some embodiments, the cancer is testicular cancer. Insome embodiments, the cancer is uterine carcinoma.

In some embodiments, the cancer comprises a glutamine-dependent cancerin which glutamine is an important fuel source for cellular energy inthe cancer (e.g., hematopoietic tumors, hepatomas, Ehrilich carcinoma(see Huber et al., “Uptake of glutamine antimetabolites6-diazo-5-oxo-L-norleucine (DON) in sensitive and resistant tumor celllines,” Int. J. Cancer. 1988; 41:752-755)).

In some embodiments, the cancer is a Myc-dependent cancer. As usedherein, “Myc-dependent cancer” refers to a cancer exhibiting activation,overexpression and/or amplification of c-Myc. In some contexts, a“Myc-dependent cancer” is a cancer in which c-Myc plays a role inincreased glutamine metabolism in the cancer cells. Examples ofMyc-dependent cancers include, without limitation, lymphoma,neuroblastoma, and small cell lung cancer.

In some embodiments, the cancer is an mTORC1-dependent cancer. As usedherein, “mTORC1-dependent cancer” refers to a cancer in which mTORC1 isactivated in a glutamine-dependent manner, i.e., that is mTORC1 plays arole in increased glutamine metabolism in the cancer cells.

The presently disclosed subject matter contemplates using a prodrug offormula (I), or a pharmaceutical composition comprising the prodrug offormula (I), optionally together with at least one immunosuppressantand/or anti-inflammatory agent, to treat an autoimmune disease, immunedisorder, or inflammatory disorder.

As used herein, “immunosuppressant agent” means an agent which may beused in immunotherapy to reduce or prevent an immune response in a cell,organ, tissue, or subject. Examples of immunosuppressant agentscontemplated for use in combination with a prodrug of formula (I), or apharmaceutical composition comprising a prodrug of formula (I) includecorticosteriods, calcineurin inhibitors, antiproliferative agents, SIPreceptor agonists, kinase inhibitors, monoclonal antilymphocyteantibodies and polyclonal antilymphocyte antibodies. Non-limitingexamples of corticosteroids include Prednisone (Deltasone® and Orasone®)and Methylprednisolone (SoluMedrol®). Non-limiting examples ofcalcineurin inhibitors include Cyclosporine (Cyclosporin A, SangCya,Sandimmune®, Neoral®, Gengraf®), ISA, Tx247, ABT-281, ASM 981 andTacrolimus (Prograf®, FK506). Non-limiting examples of antiproliferativeagents include Mycophenolate Mofetil (CellCept®), Azathioprene(Imuran®), and Sirolimus (Rapamune®). Non-limiting examples of SIPreceptor agonists include FTY 720 or analogues thereof. Non-limitingexamples of kinase inhibitors include mTOR kinase inhibitors, which arecompounds, proteins or antibodies that target, decrease or inhibit theactivity and/or function of members of the serine/threonine mTOR family.These include, without limitation, CCI-779, ABT578, SAR543, rapamycinand derivatives or analogs thereof, including40-O-(2-hydroxyethyl)-rapamycin, rapalogs, including AP23573, AP23464,AP23675 and AP23841 from Ariad, Everolimus (CERTICAN, RAD001), biolimus7, biolimus 9 and sirolimus (RAPAMUNE). Kinase inhibitors also includeprotein kinase C inhibitors, which include the compounds described thePCT publications WO 2005/097108 and WO 2005/068455, which are hereinincorporated by reference in their entireties. Non-limiting examples ofmonoclonal antilymphocyte antibodies include Muromonab-CD3 (OrthocloneOKT3®), Interleukin-2 Receptor Antagonist (Basiliximab, Simulect®), andDaclizumab (Zenapax®). Non-limiting examples of polyclonalantilymphocyte antibodies include Antithymocyte globulin-equine (Atgam®)and Antithymocyte globulin-rabbit (RATG, Thymoglobulin®). Otherimmunosuppressants include, without limitation, SERP-1, a serineprotease inhibitor produced by malignant rabbit fibroma virus (MRV) andmyxoma virus (MYX), described in US Patent Publication No. 2004/0029801,which is incorporated herein by reference.

As used herein, “anti-inflammatory agent” refers to an agent that may beused to prevent or reduce an inflammatory response or inflammation in acell, tissue, organ, or subject. Exemplary anti-inflammatory agentscontemplated for use in combination with a prodrug of formula (I), or apharmaceutical composition comprising a prodrug of formula (I) include,without limitation, steroidal anti-inflammatory agents, a nonsteroidalanti-inflammatory agent, or a combination thereof. In some embodiments,anti-inflammatory agents include clobetasol, alclofenac, alclometasonedipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide,amfenac sodium, amiprilose hydrochloride, anakinra, anirolac,anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen,benzydamine hydrochloride, bromelains, broperamole, budesonide,carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate,clobetasone butyrate, clopirac, cloticasone propionate, cormethasoneacetate, cortodoxone, deflazacort, desonide, desoximetasone,dexamethasone, dexamethasone acetate, dexamethasone dipropionate,diclofenac potassium, diclofenac sodium, diflorasone diacetate,diflumidone sodium, diflunisal, difluprednate, diftalone, dimethylsulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium,epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen,fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone,fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin,flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, methylprednisolone suleptanate,momiflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone,olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone,paranyline hydrochloride, pentosan polysulfate sodium, phenbutazonesodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicamolamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone,proxazole, proxazole citrate, rimexolone, romazarit, salcolex,salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin,sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate,tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide,tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium,triclonide, triflumidate, zidometacin, zomepirac sodium, aspirin(acetylsalicylic acid), salicylic acid, corticosteroids,glucocorticoids, tacrolimus, pimecorlimus, prodrugs thereof, co-drugsthereof, and combinations thereof. The anti-inflammatory agent may alsobe a biological inhibitor of proinflammatory signaling moleculesincluding antibodies to such biological inflammatory signalingmolecules.

Autoimmunity is the failure of an organism to recognize its ownconstituent parts (down to the sub-molecular levels) as “self,” whichresults in an immune response against its own cells and tissues. Anydisease that results from such an aberrant immune response is termed anautoimmune disease. An unwanted immune response may be, for example,immune responses associated with an autoimmune disorder, allergies, orinflammatory disorders. The term “immune disorders” are diseasesinvolving the immune system that can include but not be limited toallergies, autoimmune diseases, immune complex diseases,immunodeficiency diseases and cancers of the immune system. In someembodiments, the autoimmune disease, immune disorder, or inflammatorydisorder is multiple sclerosis.

The presently disclosed subject matter contemplates using a prodrug offormula (I), or a pharmaceutical composition comprising the prodrug offormula (I), optionally together with at least one neuroprotective agentand/or at least one neurotrophic factor, and/or at least oneneuroregenerative agent, to treat a neurodegenerative or neurologicaldisorder or disease.

A “neurodegenerative disorder” is a disease, disorder, or condition thatis characterized by the progressive loss of the structure or function ofneurons (e.g., degeneration or dysfunction of neurons or other neuralcells). Glutaminase-catalyzed hydrolysis of glutamine to glutamate is apredominant source of brain glutamate. Normal central nervous system(CNS) synaptic transmission uses glutamate as the major excitatory aminoacid neurotransmitter. Excessive glutamatergic signaling, known asexcitotoxicity, is believed to cause CNS damage in variousneurodegenerative diseases, such as stroke, amyotrophic lateralsclerosis (ALS), Huntington's disease, Alzheimer's disease, andHIV-associated dementia. Accordingly, without wishing to be bound bytheory, it is believed that the presently disclosed prodrugs can be usedto treat such neurodegenerative diseases, for example, by inhibitingglutaminase to ameliorate the CNS damage resulting from excitotoxicitydue to excessive glutamatergic signaling.

In particular embodiments, the neurodegenerative disorder is multiplesclerosis (MS). DON has been shown to be effective in amelioratingexperimental autoimmune enchaphalomyelitis (EAE), an animal model ofmultiple sclerosis (MS) (see, e.g., Shijie, et al., “Blockade ofglutamate release from microglia attenuates experimental autoimmuneencephalomyelitis in mice,” Tohoku J. Exp. Med. 2009; 217:87-92). Inparticular embodiments, the neurodegenerative disorder is HIV-associateddementia (HAD). In particular embodiments, the neurodegenerativedisorder is ischemia (e.g., transient ischemic brain injury). Inparticular embodiments, the neurodegenerative disorder is stroke. Inparticular embodiments, the neurodegenerative disorder is amyotrophiclateral sclerosis (ALS). In particular embodiments, theneurodegenerative disorder is Huntington's disease. In particularembodiments, the neurodegenerative disorder is Alzheimer's disease.

In some embodiments, the presently disclosed subject matter providesmethods for inhibiting the excess and/or aberrant glutamine activityfound in a subject with a disease or condition. As used herein, the term“inhibit” means to decrease or diminish the excess and/or aberrantglutamine activity found in a subject. The term “inhibit” also may meanto decrease, suppress, attenuate, diminish, arrest, or stabilize thedevelopment or progression of a disease or condition. Inhibition mayoccur, for e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or even 100% compared to an untreated controlsubject or a subject without the disease or disorder. As used herein,the term “excess glutamine activity” means an increase in glutamineactivity in a subject with a disease or condition as compared to theglutamine activity in a subject without a similar disease or condition,such as an increase of approximately 100%, 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900%, 1000%, or more. As used herein, the term“aberrant glutamine activity” means a change in the biological activityof glutamine in a subject with a disease or condition as compared to theglutamine activity in a subject without a similar disease or condition,such utilization of glutamine in the growth and/or proliferation ofmalignant, neoplastic, or other pathologic cellular processes.

In some embodiments, the disease or condition involves excess and/oraberrant glutamine activity. In such aspects, the method furthercomprises inhibiting the excess and/or aberrant glutamine activity whenthe compound of formula (I), or the pharmaceutical composition of anythereof, is administered.

In another aspect, the presently discloses subject matter involves theuse of a compound of formula (I), or a pharmaceutical composition of anythereof, for treating a disease or condition. In some embodiments, thecompound of formula (I), or the pharmaceutical composition of anythereof is used to treat a disease or condition selected from the groupconsisting of an infection, cancer, an autoimmune disease, aninflammatory disease, and a neurodegenerative or neurological disease.In some embodiments, the compound of formula (I), or the pharmaceuticalcomposition of any thereof is used to treat a disease or conditionselected from the group consisting of multiple sclerosis, convulsions,epilepsy, and viral encephalitis. In some embodiments, the compound offormula (I), or the pharmaceutical composition of any thereof is used totreat a disease or condition that involves excess and/or aberrantglutamine activity. In such aspects, the use involves inhibiting theexcess and/or aberrant glutamine activity when the compound of formula(I), or the pharmaceutical composition of any thereof, is used to treatthe disease or condition.

IV. General Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

While the following terms in relation to compounds of formula (I) arebelieved to be well understood by one of ordinary skill in the art, thefollowing definitions are set forth to facilitate explanation of thepresently disclosed subject matter. These definitions are intended tosupplement and illustrate, not preclude, the definitions that would beapparent to one of ordinary skill in the art upon review of the presentdisclosure.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group on a molecule, provided that the valency of all atomsis maintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents also may be further substituted (e.g., anaryl group substituent may have another substituent off it, such asanother aryl group, which is further substituted at one or morepositions).

Where substituent groups or linking groups are specified by theirconventional chemical formulae, written from left to right, they equallyencompass the chemically identical substituents that would result fromwriting the structure from right to left, e.g., —CH₂O— is equivalent to—OCH₂—; —C(═O)O— is equivalent to —OC(═O)—; —OC(═O)NR— is equivalent to—NRC(═O)O—, and the like.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₁, R₂, and the like, orvariables, such as “m” and “n”), can be identical or different. Forexample, both R₁ and R₂ can be substituted alkyls, or R₁ can be hydrogenand R₂ can be a substituted alkyl, and the like.

The terms “a,” “an,” or “a(n),” when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

A named “R” or group will generally have the structure that isrecognized in the art as corresponding to a group having that name,unless specified otherwise herein. For the purposes of illustration,certain representative “R” groups as set forth above are defined below.

Descriptions of compounds of the present disclosure are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless otherwise explicitly defined, a “substituent group,” as usedherein, includes a functional group selected from one or more of thefollowing moieties, which are defined herein:

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. As would be known to one skilled in this art, allvalencies must be satisfied in making any substitutions. The hydrocarbonmay be unsaturated, saturated, branched, unbranched, cyclic, polycyclic,or heterocyclic. Illustrative hydrocarbons are further defined hereinbelow and include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, andthe like.

Further, more generally, a “carbyl” refers to a carbon atom or a moietycomprising one or more carbon atoms acting as a bivalent radical.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, acyclic or cyclic hydrocarbon group, or combination thereof,which may be fully saturated, mono- or polyunsaturated and can includedi- and multivalent groups, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7,8, 9, and 10 carbons). In particular embodiments, the term “alkyl”refers to C₁₋₂₀ inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containingbetween one and twenty carbon atoms by removal of a single hydrogenatom.

Representative saturated hydrocarbon groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl,sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂₅—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to twoor three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

As described above, heteroalkyl groups, as used herein, include thosegroups that are attached to the remainder of the molecule through aheteroatom, such as —C(O)NR′, —NR′R″, —OR′, —SR, —S(O)R, and/or—S(O₂)R′. Where “heteroalkyl” is recited, followed by recitations ofspecific heteroalkyl groups, such as —NR′R or the like, it will beunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity. Thus, the term “heteroalkyl” should not be interpretedherein as excluding specific heteroalkyl groups, such as —NR′R″ or thelike.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, unsubstituted alkyl,substituted alkyl, aryl, or substituted aryl, thus providing aheterocyclic group. Representative monocyclic cycloalkyl rings includecyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl ringsinclude adamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl, and fused ring systems, such as dihydro- andtetrahydronaphthalene, and the like.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, unsaturated or partially unsaturated ringsystem, such as a 3- to 10-member substituted or unsubstitutedcycloalkyl ring system, including one or more heteroatoms, which can bethe same or different, and are selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si),and optionally can include one or more double bonds.

The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or a polycyclic group wherein at least one ring atom isa heteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), including, but not limited to,a bi- or tri-cyclic group, comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from theoxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to an aryl or heteroaryl ring. Representativecycloheteroalkyl ring systems include, but are not limited topyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl,morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and thelike.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

An unsaturated alkyl group is one having one or more double bonds ortriple bonds. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl.”

More particularly, the term “alkenyl” as used herein refers to amonovalent group derived from a C₁₋₂₀ inclusive straight or branchedhydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen molecule. Alkenyl groups include, forexample, ethenyl (i.e., vinyl), propenyl, butenyl,1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, allenyl, andbutadienyl.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarboncontaining at least one carbon-carbon double bond. Examples ofcycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a monovalent group derivedfrom a straight or branched C₁₋₂₀ hydrocarbon of a designed number ofcarbon atoms containing at least one carbon-carbon triple bond. Examplesof “alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl,pentynyl, hexynyl, and heptynyl groups, and the like.

The term “alkylene” by itself or a part of another substituent refers toa straight or branched bivalent aliphatic hydrocarbon group derived froman alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—,—CH₂CsCCH₂—, —CH₂CH₂CH(CH₂CH₂CH₃)CH₂—, —(CH₂)_(q)—N(R)—(CH₂)_(r)—,wherein each of q and r is independently an integer from 0 to about 20,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl(—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group canhave about 2 to about 3 carbon atoms and can further have 6-20 carbons.Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being someembodiments of the present disclosure. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent group derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms also can occupy either or both of thechain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)OR′— represents both —C(O)OR′—and —R′OC(O)—.

The term “aryl” means, unless otherwise stated, an aromatic hydrocarbonsubstituent that can be a single ring or multiple rings (such as from 1to 3 rings), which are fused together or linked covalently. Arylsinclude phenyl (C₆), naphthyl (C₁₀), and biphenyl (C₁₂).

The term “heteroaryl” refers to aryl groups (or rings) that contain fromone to four heteroatoms (in each separate ring in the case of multiplerings) selected from N, O, and S, wherein the nitrogen and sulfur atomsare optionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroaryl group can be attached to the remainder of themolecule through a carbon or heteroatom. Non-limiting examples of aryland heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent forms of aryl and heteroaryl, respectively.

In further embodiments, the term “heteroaryl” refers to a C₅-C₂₀aromatic ring wherein at least one carbon atom is replaced by aheteroatom selected from O, S, N, optionally substituted by at least onesubstituent selected from the group consisting of C₆ alkyl, hydroxy,C₁-C₄ alkoxy, mercapto, C₁-C₄ alkylthio, amino, —NH(C₁-C₆ alkyl),—N(C₁-C₆ alkyl)₂, —O(C₆-C₁₂ aryl), —N(C₆-C₁₂ aryl)₂, —NH(C₆-C₁₂ aryl),—S(C₆-C₁₂ aryl), halogen, —CF₃, —SO₃H, —COOH, —COO(C₁-C₈ alkyl),—SO₂NH₂, —SO₂NH(C₁-C₆ alkyl or C₆-C₁₂ aryl), —CN, —NO₃, —C(O)(C₁-C₈alkyl), —C(O)(C₆-C₁₂ aryl), —N(C₁-C₆ alkyl or H)C(O)(C₁-C₆ alkyl or H),—C(O)N(C₁-C₆ alkyl or H)₂.

Exemplary heteroaryls include, but are not limited to, pyridyl,pyrimidinyl, pyrazinyl, furanyl, thienyl, pyrazolyl, oxazolyl,thiazolyl, isothiazolyl, isoxazolyl, pyrrolyl, imidazolyl, indolyl,indolinolyl, and imidazopyridazinyl.

In further embodiments, the term “aryl” also can refer to C₆-C₁₄ aryl,optionally substituted by at least one substituent selected from thegroup consisting of C₁-C₆ alkyl, hydroxy, C₁-C₄ alkoxy, mercapto, C₁-C₄alkylthio, amino, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —O(C₆-C₁₂ aryl),—N(C₆-C₁₂ aryl)₂, —NH(C₆-C₁₂ aryl), —S(C₆-C₁₂ aryl), halogen, —CF₃,—SO₃H, —COOH, —COO(C₁-C₈ alkyl), —SO₂NH₂, —SO₂NH(C₁-C₆ alkyl or C₆-C₁₂aryl), —CN, —NO₃, —C(O)(C₁-C₈ alkyl), —C(O)(C₆-C₁₂ aryl), —N(C₁-C₆ alkylor H)C(O)(C₁-C₆ alkyl or H), —C(O)N(C₁-C₆ alkyl or H)₂.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the terms “arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl orheteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl, furylmethyl, and the like) including those alkyl groupsin which a carbon atom (e.g., a methylene group) has been replaced by,for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like). However, the term “haloaryl,” asused herein is meant to cover only aryls substituted with one or morehalogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g., “3 to 7 membered”), the term “member” refers toa carbon or heteroatom. Further, a structure represented generally bythe formula:

as used herein refers to a ring structure, for example, but not limitedto a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and thelike, aliphatic and/or aromatic cyclic compound, including a saturatedring structure, a partially saturated ring structure, and an unsaturatedring structure, comprising a substituent R group, wherein the R groupcan be present or absent, and when present, one or more R groups caneach be substituted on one or more available carbon atoms of the ringstructure. The presence or absence of the R group and number of R groupsis determined by the value of the variable “n,” which is an integergenerally having a value ranging from 0 to the number of carbon atoms onthe ring available for substitution. Each R group, if more than one, issubstituted on an available carbon of the ring structure rather than onanother R group. For example, the structure above where n is 0 to 2would comprise compound groups including, but not limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is, adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

The symbol (

)denotes the point of attachment of a moiety to the remainder of themolecule.

When a named atom of an aromatic ring or a heterocyclic aromatic ring isdefined as being “absent,” the named atom is replaced by a direct bond.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl,” “aryl,” “heteroaryl,” “phosphonate,” and “sulfonate”as well as their divalent derivatives) are meant to include bothsubstituted and unsubstituted forms of the indicated group. Optionalsubstituents for each type of group are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative groups (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′, —NR—C(NR′R″)═NR′″, —S(O)R′,(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a number ranging fromzero to (2m′+1), where m′ is the total number of carbon atoms in suchgroups. R′, R″, R′″ and R″″ each may independently refer to hydrogen,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. As used herein, an “alkoxy” group is an alkyl attachedto the remainder of the molecule through a divalent oxygen. When acompound of the disclosure includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. When R′and R″ are attached to the same nitrogen atom, they can be combined withthe nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl groups above, exemplarysubstituents for aryl and heteroaryl groups (as well as their divalentderivatives) are varied and are selected from, for example: halogen,—OR′, —NR′R″, —SR′, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″R′″)═NR′″, —NR—C(NR′R″)═NR′″—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxo, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on aromatic ring system; and where R′, R″, R′″ and R″″ maybe independently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the disclosure includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4.

One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where sand d are independently integers of from 0 to 3, and X′ is —O—, —NR′—,—S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R′″may be independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituentand has the general formula RC(═O)—, wherein R is an alkyl, alkenyl,alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic groupas defined herein). As such, the term “acyl” specifically includesarylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetylgroup. Specific examples of acyl groups include acetyl and benzoyl. Acylgroups also are intended to include amides, —RC(═O)NR′, esters,—RC(═O)OR′, ketones, —RC(═O)R′, and aldehydes, —RC(═O)H.

The terms “alkoxyl” or “alkoxy” are used interchangeably herein andrefer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O—and alkynyl-O—) group attached to the parent molecular moiety through anoxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are aspreviously described and can include C₁₋₂₀ inclusive, linear, branched,or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including,for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl,sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, andthe like.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl,i.e., C₆H₅—CH₂—O—. An aralkyloxyl group can optionally be substituted.

“Alkoxycarbonyl” refers to an alkyl-O—C(═O)— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and tert-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—C(═O)— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—C(═O)— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an amide group of the formula —C(═O)NH₂.“Alkylcarbamoyl” refers to a R′RN—C(═O)— group wherein one of R and R′is hydrogen and the other of R and R′ is alkyl and/or substituted alkylas previously described. “Dialkylcarbamoyl” refers to a R′RN—C(═O)—group wherein each of R and R′ is independently alkyl and/or substitutedalkyl as previously described.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula —O—C(═O)—OR.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group and also refers to a nitrogencontaining group as is known in the art derived from ammonia by thereplacement of one or more hydrogen radicals by organic radicals. Forexample, the terms “acylamino” and “alkylamino” refer to specificN-substituted organic radicals with acyl and alkyl substituent groupsrespectively.

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. More particularly, the terms alkylamino,dialkylamino, and trialkylamino as used herein refer to one, two, orthree, respectively, alkyl groups, as previously defined, attached tothe parent molecular moiety through a nitrogen atom. The term alkylaminorefers to a group having the structure —NHR′ wherein R′ is an alkylgroup, as previously defined; whereas the term dialkylamino refers to agroup having the structure —NR′R″, wherein R′ and R″ are eachindependently selected from the group consisting of alkyl groups. Theterm trialkylamino refers to a group having the structure —NR′R″R′″,wherein R′, R″, and R″ are each independently selected from the groupconsisting of alkyl groups. Additionally, R′, R″, and/or R″ takentogether may optionally be —(CH₂)_(k)— where k is an integer from 2 to6. Examples include, but are not limited to, methylamino, dimethylamino,ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino,isopropylamino, piperidino, trimethylamino, and propylamino.

The amino group is —NR′R″, wherein R′ and R″ are typically selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e.,alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Examplesof thioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is aspreviously described.

The term “carbonyl” refers to the —C(═O)— group, and can include analdehyde group represented by the general formula R—C(═O)H.

The term “carboxyl” refers to the —COOH group. Such groups also arereferred to herein as a “carboxylic acid” moiety.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups. Additionally, terms, such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. Forexample, the term “halo(C₁-C₄)alkyl” is mean to include, but not belimited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” as used herein means an oxygen atom that is double bondedto a carbon atom or to another element.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

The term thiohydroxyl or thiol, as used herein, refers to a group of theformula —SH.

More particularly, the term “sulfide” refers to compound having a groupof the formula —SR.

The term “sulfone” refers to compound having a sulfonyl group —S(O₂)R.

The term “sulfoxide” refers to a compound having a sulfinyl group —S(O)R

The term ureido refers to a urea group of the formula —NH—CO—NH₂.

Throughout the specification and claims, a given chemical formula orname shall encompass all tautomers, congeners, and optical- andstereoisomers, as well as racemic mixtures where such isomers andmixtures exist.

Certain compounds of the present disclosure may possess asymmetriccarbon atoms (optical or chiral centers) or double bonds; theenantiomers, racemates, diastereomers, tautomers, geometric isomers,stereoisometric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as D- or L- for amino acids, andindividual isomers are encompassed within the scope of the presentdisclosure. The compounds of the present disclosure do not include thosewhich are known in art to be too unstable to synthesize and/or isolate.The present disclosure is meant to include compounds in racemic,scalemic, and optically pure forms. Optically active (R)- and (S)-, orD- and L-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. When the compoundsdescribed herein contain olefenic bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structuralisomers which exist in equilibrium and which are readily converted fromone isomeric form to another.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures with the replacement of a hydrogen by a deuterium or tritium,or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are withinthe scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present disclosure, whether radioactive or not, are encompassedwithin the scope of the present disclosure.

The compounds of the present disclosure may exist as salts. The presentdisclosure includes such salts. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.,(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures, succinates, benzoates and salts with amino acids, such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. Also included are base addition salts, such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present disclosure containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent or byion exchange. Examples of acceptable acid addition salts include thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids, such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

In addition to salt forms, the present disclosure provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the compounds ofthe present disclosure by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present disclosure when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” may not expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but maybe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments, ±100% insome embodiments ±50%, in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Methods

General Procedures:

The commercially available HPLC grade methanol, catalysts and reagentgrade materials were used as received. TLC was performed on Silica gel60 F254-coated aluminum sheets (Merck) and spots were detected by thesolution of Ce(SO₄)₂. 4H₂O (1%) and H₃P(Mo₃O₁₀)₄ (2%) in sulfuric acid(10%). Flash chromatography was performed on Silica gel 60 (0.040-0.063mm, Fluka) or on Biotage® KP-C18-HS or KP-Sil® SNAP cartridges using theIsolera One HPFC system (Biotage, Inc.). All chemicals were purchasedfrom Sigma-Aldrich and were used without further purification. The ¹HNMR spectra were measured at 400.1 MHz, 500.1 MHz or 600.1 MHz, ¹³C NMRspectra at 100.8 MHz, 125.7 MHz or 150.9 MHz. For standardization of ¹HNMR spectra the internal signal of TMS (δ 0.0, CDCl₃) or residualsignals of solvents (δ 7.26 for CDCl₃, δ 2.05 for CD₃COCD₃ and δ 3.31for CD₃OD) were used. In the case of ¹³C spectra the residual signals ofsolvents (δ 77.00 for CDCl₃, δ 29.84 and δ 206.26 for CD₃COCD₃ and δ49.00 for CD₃OD) were used. The chemical shifts are given in 6-scale,the coupling constants J are given in Hz. The ESI mass spectra wererecorded using ZQ micromass mass spectrometer (Waters) equipped with anESCi multimode ion source and controlled by MassLynx software.Alternatively, the low resolution ESI mass spectra were recorded using aquadrupole orthogonal acceleration time-of-flight tandem massspectrometer (Q-Tof micro, Waters) and high resolution ESI mass spectrausing a hybrid FT mass spectrometer combining a linear ion trap MS andthe Orbitrap mass analyzer (LTQ Orbitrap XL, Thermo Fisher Scientific).The conditions were optimized for suitable ionization in the ESIOrbitrap source (sheat gas flow rate 35 a.u., aux gas flow rate 10 a.u.of nitrogen, source voltage 4.3 kV, capillary voltage 40 V, capillarytemperature 275° C., tube lens voltage 155 V). The samples weredissolved in methanol and applied by direct injection. Optical rotationswere measured in CHCl₃ or DMF using an Autopol IV instrument (RudolphResearch Analytical). The IR spectra were measured in CHCl₃ or KBr.

Mice Efficacy Studies.

All mouse efficacy studies were conducted according to protocol #M013M69approved by the Animal Care and Use Committee at Johns HopkinsUniversity. Female athymic (RH-Foxnlnu mice) mice between 25 and 30 gwere obtained (Harlan Sprague Dawley Inc, Indianapolis, Ind.), andmaintained on a 12 hour light-dark cycle with ad libitum access to foodand water. U87 human glioma cells were injected s.c. (5×10⁶ cells in 100ml of PBS) in four separate locations on the flanks of each mouse. Whentumors grew to a mean volume of around 200 mm³, mice were randomizedinto either vehicle (HEPES-buffered saline, i.p.) or DON (1; 0.8 mg/kg,i.p.). In one cohort, mice were administered a single dose of theappropriate solution two hours after which glutamine levels werequantified in the tumor as described previously (Le et al., 2012). Inbrief, tumors were harvested, snap frozen, and homogenized in liquid N₂then subjected to metabolite extraction using methanol and DI water.Quantification was performed using Agilent 6520Quadrupole-Time-of-Flight (Q-TOF) mass spectrometer with Agilent 1290HPLC and using Agilent Mass Hunter and Agilent Qualitative andQuantitative Analysis Software packages. Glutamine content was averagedby group for each individual tumor (n=3-4/group), depicted as relativeintensity, and analyzed by one-tailed t test. In a second cohort,efficacy experiments were conducted. Mice were injected once daily forsix days; tumor volumes were measured using digital calipers andcalculated according to the formula: [volume=(largest tumordimension)×(smallest tumor dimension)²×0.52] at 2, 4, and 6 days afterthe onset of treatment. Each individual tumor (n=8-10/group) wasnormalized to its pretreatment volume, averaged and analyzed by repeatedmeasures two-way analysis of variance (ANOVA). If significant, aBonferroni post hoc test was subsequently applied. Significance wasdefined as p<0.05.

In Vitro Stability Studies:

The stock solution for most prodrugs was prepared as a 10 mM solution inDMSO to carry out the in vitro studies.

The chemical stability of prodrugs was evaluated using simulated gastricfluid (SGF; pH 1.2) and phosphate buffered saline (PBS; pH 7.4).Briefly, prodrugs were spiked (10 μM) in respective solutions andincubated at 37° C. for 1 h. At predetermined time points (0, 30 and 60min), aliquots of 100 μL were removed and diluted with 100 μL of water.Prodrug disappearance was monitored using the developed liquidchromatography and tandem mass spectrometry (LC/MS/MS) method describedbelow.

For metabolic stability, plasma (dog, human, monkey, mouse and pig andhuman) was used. For stability, prodrugs (10 μM) were spiked in eachmatrix and incubated in an orbital shaker at 37° C. At predeterminedtimes (0, 30 and 60 min), 100 μL aliquots of the mixture in triplicatewere removed and the reaction quenched by addition of three times thevolume of ice cold acetonitrile spiked with the internal standard(losartan 0.5 μM). The samples were vortexed for 30 s and centrifuged12000 g for 10 min. 50 μL supernatant diluted with 50 μL water wastransferred to a 250 μL polypropylene vial sealed with a Teflon cap.Prodrug disappearance was monitored over time using a liquidchromatography and tandem mass spectrometry (LC/MS/MS) method asdescribed below.

For LC/MS/MS, prodrugs were separated with Thermo Scientific Accela UPLCsystem coupled to Accela open autosampler on an Agilent C18 (100×2.1 mmid) UPLC column. The autosampler was temperature controlled andoperating at 10° C. The mobile phase used for the chromatographicseparation was composed of acetonitrile/water containing 0.1% formicacid and was run at a flow rate of 0.5 mL/minute for 4.5 minutes usinggradient elution. The column effluent was monitored using TSQ Vantagetriple-quadrupole mass-spectrometric detector, equipped with anelectrospray probe set in the positive ionization mode. Samples wereintroduced into the ionization source through a heated nebulized probe(350° C.).

For quantification of compound remaining, disappearance of prodrugs wasmeasured from ratio of peak areas of analyte to IS. Percentage remainingwas calculated in the following manner:

$\frac{{{Avg}.\mspace{14mu}{Response}}*{@60}\mspace{14mu}\min}{{{Avg}.\mspace{14mu}{{Response}\mspace{14mu}@0}}\mspace{14mu}\min} \times 100$where response=[(Area of analyte)/(Area of internal standard)]*Average response is average of two samples at each time point.

Pharmacokinetic Studies in Mice.

All pharmacokinetic studies in mice were conducted according to protocol(#M013M113) approved by the Animal Care and Use Committee at JohnsHopkins University. C57BL/6 mice between 25 and 30 g were obtained fromHarlan, and maintained on a 12 hour light-dark cycle with ad libitumaccess to food and water. To evaluate the brain and plasmapharmacokinetics of DON and its prodrug 5c, 8-12 week old C57BL/6 wereadministered DON (1; 0.8 mg/kg, p.o. in phosphate-buffered saline) andits prodrug 5c (at 0.8 mg/kg equivalent DON (1), p.o. inphosphate-buffered saline with 5% EtOH and 5% Tween-80). The mice weresacrificed by pentobarbital injection at 10, 30 and 90 minutes post drugadministration, and blood was collected via cardiac puncture and placedinto iced EDTA coated BD microtainers. Blood samples were spun at 2,000g for 15 minutes, and plasma was removed and stored at −80° C. Braintissues were harvested following blood collection and immediately snapfrozen in liquid nitrogen and stored at −80° C. until LC/MS analysis.

Pharmacokinetic Studies in Non-Human Primates.

All monkey studies were conducted according to protocol (#PR15M298)approved by the Animal Care and Use Committee at Johns HopkinsUniversity. Two female pigtail monkeys (approximately 3.5 kg, non-drugnaive) were adjacently housed in stainless steel cages on a socialinteraction rack (contains 4 cages, each 32.5″ wide×28″ deep×32″ high)maintaining temperature of 64-84° F., humidity of 30-70% withalternating 14-10 hour light/dark cycle as per the USDA Animal WelfareAct (9 CFR, Parts 1, 2, and 3). Food was provided daily in amountsappropriate for the size and age of the animals and RO purified waterprovided ad libitum through an in-cage lixit valve. Food enrichment wasprovided Monday through Friday. Prior to drug administration, macaqueswere sedated with ketamine given as an intramuscular injection prior totest article administration. Sedation was maintained through blood andcerebrospinal fluid (CSF) sample collections with ketamine at a startingrate of 15 mg/kg with additional doses of 20-30 mg during the firsthour. At subsequent time points ketamine was given at 10-15 mg/kg. DON(50 mM HEPES buffered saline) and 5c (Diastereoisomer 1), (50 mM HEPESbuffered saline containing 5% ethanol and 5% tween) were administered(1.6 mg/kg equivalent) to the animals at a dosing volume of 1 mL/kgintravenously. CSF sample (target of 50 μL) was obtained by percutaneouspuncture of the cisterna magna at 30 min post dose. Blood samples (1 mL)were collected at 15, 30, 1, 2 4 and 6 h post dose by percutaneouspuncture of a peripheral vein. Samples were processed for plasma(centrifuged at a temperature of 4° C., at 3,000×g, for 10 minutes). Allsamples were maintained chilled on ice throughout processing. Sampleswere collected in microcentrifuge tubes, flash frozen, and placed in afreezer set to maintain −80° C. until LC/MS analysis.

Bioanalysis of DON.

A highly sensitive method for analysis of DON in biological matrices(Alt, et al., 2015) has been previously published. However due tochemical lability of DON and its prodrugs, a milder derivatizationmethod employing dabsyl chloride was developed and validated. Briefly,DON was extracted from samples (50 mg) with 250 μL methanol containingGlutamate-d5 (10 μM ISTD) by vortexing in low retention tubes. Sampleswere centrifuged at 16,000×g for 5 minutes to precipitate proteins.Supernatants (200 μL) were moved to new tube and dried at 45° C. undervacuum for 1 hour. To each tube, 50 μL of 0.2 M sodium bicarbonatebuffer (pH 9.0) and 100 μL of 10 mM dabsyl chloride in acetone wasadded. After vortexing, samples were incubated at 60° C. for 15 minutesto derivatize. Samples (2 μL) were injected and separated on an Agilent1290 equipped with a an Agilent Eclipse plus C18 RRHD 2.1 X100 mm columnover a 2.5 minute gradient from 20-95% acetonitrile+0.1% formic acid andquantified on an Agilent 6520 QTOF mass spectrometer. Calibration curvesover the range of 0.005-17.1 μg/mL in plasma and CSF for DON wereconstructed from the peak area ratio of the analyte to the internalstandard using linear regression with a weighting factor of 1/(nominalconcentration). Correlation coefficient of greater than 0.99 wasobtained in all analytical runs. The mean predicted relative standarddeviation for back calculated concentrations of the standards and QC'sfor all analytes were within the range of 85 to 115%, except for thelowest concentration which was within the range of 80 to 120% with anoverall accuracy and precision of 6.7% and 6.6%, respectively.

Pharmacokinetic Analysis.

Mean concentration-time data were used for pharmacokinetic analysis.Non-compartmental-analysis module in WinNonlin® (version 5.3) was usedto assess pharmacokinetic parameters. Peak plasma concentrations(C_(max)) and time to C_(max) (T_(max)) were the observed values. Areaunder the curve (AUC) was calculated by log-linear trapezoidal rule tothe end of sample collection (AUC_(last)).

Procedure for Pharmacokinetic analysis for DON release from itsprodrugs: DON is extracted from samples (50 μL) with 250 μL methanolcontaining Glutamate-d5 (10 μM ISTD) by vortexing in low retentiontubes. Samples are centrifuged at 16,000×g for 5 minutes to precipitateproteins. Supernatants (200 μL) are moved to new tube and dried at 45°C. under vacuum (approximately 1 hour). To each tube, 50 μL of 0.2 Msodium bicarbonate buffer (pH 9.0) and 100 μL dabsyl chloride stock isadded. After vortexing, samples are incubated at 60° C. for 15 minutesto derivatize. Samples (2-10 μL) are injected and separated on anAgilent 1290 equipped with a SB-AQ column over a 4 minute gradient from20-95% acetonitrile+0.1% formic acid and quantified on an Agilent 6520QTOF mass spectrometer.

Example 2 Prodrug Strategy—Masking Carboxylate Functionality

In one embodiment, DON prodrugs were designed by masking only thecarboxylate functionalities using alkyl esters of DON with unprotectedα-amino groups. Some of the alkyl esters of DON with an unprotectedα-amino functionality, however, were found to undergo cyclizationforming a 5-membered cyclic Schiff's base. The cyclization observed waspH dependent and rapid at pH 5-7. At a lower pH, which would normallyprevent or reverse the cyclization, the diazo functionality becameunstable. As a result, the cyclization was virtually irreversiblerendering some of the N-α-free alkyl esters undesirable as DON prodrugs(FIG. 1A).

Example 3 Prodrug Strategy—Masking Amino Functionality

In another embodiment, DON prodrugs were designed by masking only theamino functionalities using N-protected derivatives of Don with anunprotected carboxy functionality. The N-protected derivatives of DONwith an unprotected carboxy functionality (FIG. 1B and FIG. 1C) alsowere not as stable. More particularly, the acidic carboxy functionalitycaused gradual slow decomposition of the diazo-group. In certain saltforms, the carboxylate anion destabilizes the N-α-protecting group. Evenfurther, as is shown in FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G and FIG. 1H,many prodrugs tested with a free carboxylate (with the exception of 26)showed negligible exposure compared to DON when given orally, furthersuggesting the benefits of derivatizing both carboxylate and aminefunctionalities for oral availability.

Example 4 Synthesis

Schemes for synthesis of representative prodrug compounds are shownbelow (Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5, Scheme 6,Scheme 7, and Scheme 8).

Synthesis of Isopropyl6-diazo-2-((diphenoxyphosphoryl)amino)-5-oxohexanoate (4) 1-methylethyl5-oxoprolinate JAM0256

JAM0256 was prepared from known literature. ¹H NMR and ¹³C NMR spectrawere in agreement with the published data.

1-(9H-fluoren-9-ylmethyl) 2-(1-methylethyl)5-oxopyrrolidine-1,2-dicarboxylate (1)

Referring to scheme 1, the previous compound JAM0256 (US2008/107623 A1)(2.94 g, 17.16 mmol) was dissolved in absolute THF (90 mL) under argonand cooled to −78° C. A solution of LiHMDS (1M in hexanes, 16.3 mL,16.302 mmol, 0.95 equiv.) was added dropwise and the solution wasstirred at the same temperature for 15 min. The resultant yellow mixturewas transferred via cannula to a solution of Fmoc chloride (22.2 g, 85.8mmol, 5 equiv.) in absolute THF (90 mL) at −78° C. The reaction mixturewas stirred at −78° C. for 2 h. After this period, the reaction wasquenched with saturated NH₄Cl (100 mL). Then it was extracted with ethylacetate (3×50 mL), the combined organic layers were washed with water(40 mL), brine (40 mL) and dried over anhydrous MgSO₄. The organicsolvent was evaporated in vacuo. The residue was chromatographed onsilica gel (hexane—ethyl acetate 2:1) to afford the desired product 1(6.2 g, 92%) as a colorless solid. ¹H NMR (400 MHz, CDCl₃): 1.23 (3H, d,J=6.2), 1.26 (3H, d, J=6.3), 2.06-2.13 (1H, m), 2.34-2.45 (1H, m),2.53-2.61 (1H, m), 2.67-7.76 (1H, m), 4.31 (1H, t, J=7.5), 4.40-4.44(1H, m), 4.53-4.57 (1H, m), 4.65 (1H, dd, J=9.4, 2.6), 5.07 (1H, hept,J=6.3), 7.31-7.35 (2H, m), 7.39-7.43 (2H, m), 7.71-7.78 (4H, m). ¹³C NMR(101 MHz, CDCl₃): 21.69, 21.78, 22.01, 31.31, 46.64, 58.99, 69.20,69.78, 120.06, 120.08, 125.43, 125.57, 127.32 (2C), 127.98 (2C), 141.31,141.33, 143.39, 143.43, 151.56, 170.58, 172.92. Optical rotation: [α]²²_(D)−24.1° (c 0.332, CHCl₃). IR (CHCl₃): 3068 w, 3029 m, 2985 m, 2939 w,2883 vw, 1797 s, 1758 s, sh, 1739 vs, 1724 vs, sh, 1609 vw, 1580 vw,1479 w, 1463 m, 1452 s, 1421 w, 1386 s, 1377 m, 1305 vs, 1194 m, 1105 s,1045 m, 1033 m, 621 w, 425 w cm⁻¹. ESI MS: 416 ([M+Na]⁺). HR ESI MS:calcd for C₂₃H₂₃O₅NNa 416.14684; found 416.14694.

Isopropyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoate (2)

Referring to scheme 1, trimethylsilyl diazomethane solution (2M indiethyl ether, 6 mL, 11.93 mmol, 1.2 equiv.) was dissolved in absoluteTHF (55 mL) under argon and cooled to −98° C. A solution ofn-butyllithium (2.5 M in hexanes, 4.9 mL, 12.23 mmol, 1.23 equiv.) wasadded dropwise and the solution was stirred at −98° C. for 30 min. Theresultant mixture was transferred via cannula to a solution of previouscompound 1 (3.91 g, 9.94 mmol, 1 equiv.) in absolute THF (100 mL) at−116° C. The reaction mixture was slowly warmed to −78° C. and thenquenched with saturated NH₄Cl. Then it was extracted with ethyl acetate(3×50 mL), the combined organic layers were washed with water (40 mL),brine (40 mL) and dried over anhydrous MgSO₄. The organic solvent wasevaporated in vacuo. The residue was chromatographed on silica gel(chloroform—acetone 20:1) to afford the desired product 2 (3.68 g, 85%)as a yellowish solid. ¹H NMR (400 MHz, CDCl₃): 1.25-1.28 (6H, m),1.95-2.04 (1H, m), 2.17-2.26 (1H, m), 2.31-2.52 (2H, m), 4.22 (1H, t,J=7.1), 4.29-4.43 (3H, m), 5.06 (1H, hept, J=6.1), 5.27 (1H, s), 5.59(1H, d, J=8.2), 7.30-7.34 (2H, m), 7.38-7.42 (2H, m), 7.59-7.62 (2H, m),7.75-7.77 (2H, m). ¹³C NMR (101 MHz, CDCl₃): 21.81, 21.84, 27.69, 36.56,47.21, 53.67, 67.10, 69.62, 120.08, 120.09, 125.18, 125.21, 127.16 (2C),127.81 (2C), 141.35, 141.37, 143.75, 143.96, 156.16, 171.50, 193.67.Optical rotation: [α]²² _(D)+15.1° (c 0.674, CHCl₃). IR (CHCl₃): 3428 m,3116 w, 3068 w, 2985 m, 2940 w, 2882 w, 2110 vs, 1731 vs, sh, 1719 vs,1641 s, 1608 w, sh, 1580 vw, 1509 s, 1478 m, 1466 m, 1451 s, 1418 w, sh,1386 s, sh, 1377 s, 1349 s, 1232 s, 1105 s, 1052 s, 1033 m, 622 w, 539m, 488 m, 426 w cm⁻¹. ESI MS: 458 ([M+Na]⁺). HR ESI MS: calcd forC₂₄H₂₅O₅NaN 458.16864; found 458.16873.

Isopropyl 2-amino-6-diazo-5-oxohexanoate (3)

Referring to scheme 1, the previous compound 2 (900 mg, 2.07 mmol), wasdissolved in dichloromethane (10 mL). Piperidine (514 μL 5.17 mmol, 2.5equiv.) was added and the reaction mixture was stirred at roomtemperature for 4 h. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform—methanol 30:1) toafford the desired product (290 mg, 66%) as a yellow oil. ¹H NMR (400MHz, CDCl₃): 1.21 (3H, d, J=1.8), 1.23 (3H, d, J=1.8), 1.52 (2H, bs),1.74-1.85 (1H, m), 2.02-2.10 (1H, m), 2.36-2.53 (2H, bm), 3.37 (1H, dd,J=8.4, 5.0), 5.00 (1H, hept, J=6.3), 5.27 (1H, s). ¹³C NMR (101 MHz,CDCl₃): 21.87, 21.89, 29.65, 36.99, 53.94, 68.64, 175.21, 194.25.Optical rotation: [α]²² _(D)+6.5° (c 0.444, CHCl₃). IR (CHCl₃): 3390 w,3323 vw, 3116 w, 2984 s, 2939 m, 2877 w, 2109 vs, 1725 vs, 1640 s, 1467m, 1454 m, 1439 w, sh, 1388 s, sh, 1376 vs, 1349 s, 1199 s, 1106 vscm⁻¹. ESI MS: 236 ([M+Na]⁺); HR ESI MS: calcd for C₉H₁₅O₃N₃Na 236.1006;found 236.1007.

Isopropyl 6-diazo-2-((diphenoxyphosphoryl)amino)-5-oxohexanoate (4)

Referring to scheme 1, isopropyl DON 3 (80 mg, 0.38 mmol) was dissolvedin absolute dichloromethane (4 mL) and triethylamine (210 μL, 1.5 mmol,4 equiv.) was added. This solution was cooled to 0° C. and diphenylchlorophosphate (156 μL, 0.75 mmol, 2 equiv.) was added dropwise. Thereaction mixture was stirred at 0° C. for 30 min and then the coolingbath was removed. The reaction mixture was then stirred at roomtemperature for 2 h. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform—acetone 30:1) toafford the desired product (131 mg, 78%) as a yellow solid. ¹H NMR (400MHz, CDCl₃): 1.19 (3H, d, J=1.0), 1.21 (3H, d, J=1.0), 1.84-1.93 (1H,m), 2.07-2.16 (1H, m), 2.19-2.41 (2H, m), 3.79-3.84 (1H, m), 4.97 (1H,hept, J=6.2), 5.07 (1H, s). ¹³C NMR (101 MHz, CDCl₃): 21.75, 21.81,29.34, 35.88, 54.29, 69.80, 80.90, 120.32 (2C, d, J_(C,P)=5.0), 120.39(2C, d, J_(C,P)=4.9), 125.23 (2C, d, J_(C,P)=1.0), 125.28 (2C, d,J_(C,P)=1.0), 130.00 (2C), 150.68 (d, J_(C,P)=5.8), 150.75 (d,J_(C,P)=6.2), 171.93 (d, J_(C,P)=5.9), 193.51. ³¹P NMR (101 MHz, CDCl₃):0.32 Optical rotation: [α]²² _(D)+15.1° (c 0.337, CHCl₃). IR (CHCl₃):3383 w, 3115 w, 3101 vw, 3063 vw, 2985 m, 2938 w, 2878 vw, 2110 vs, 1731s, 1642 s, 1600 m, sh, 1591 m, 1490 s, 1467 w, 1456 m, 1448 vw, sh, 1426m, 1385 s, sh, 1377 s, 1350 m, 1191 vs, 1163 s, 1071 w, 1026 m, 941 vs,904 m, 821 w, 690 m, 617 w, 487 m cm⁻¹. ESI MS: 468 ([M+Na]⁺). HR ESIMS: calcd for C₂₁H₂₄O₆N3NaP 468.12949; found 468.12952.

Synthesis of ethyl2-((((4-acetoxybenzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate (6)1-[4-(acetyloxy)benzyl] 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (5)

To a solution of phosgene (15% wt in toluene, 10 mL, 14 mmol, 2.5equiv.) cooled to 0° C. was added a solution of 4-Acetoxybenzyl alcohol(934 mg, 5.6 mmol) in toluene (6.7 mL). The reaction mixture was stirredat the same temperature overnight. The volatiles were removed in vacuoand the product 4-(Acetoxy)benzyl Chloroformate was dissolved in THF (5mL) and used without any purification. Ethyl pyroglutamate (800 mg, 5.1mmol) was dissolved in absolute THF (13 mL) under argon and cooled to−78° C. A solution of LiHMDS (1M in hexanes, 6.12 mL, 6.12 mmol, 1.2equiv.) was added dropwise and the solution was stirred at the sametemperature for 15 min. The resultant yellow mixture was transferred viacannula to a solution of 4-(Acetoxy)benzyl chloroformate (5.6 mmol, 1equiv) at −78° C. The reaction mixture was stirred at −78° C. for 2 h.After this period, the reaction was quenched with saturated NH₄Cl (100mL). Then it was extracted with ethyl acetate (3×50 mL), the combinedorganic layers were washed with water (40 mL), brine (40 mL) and driedover anhydrous MgSO₄. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (hexane—ethyl acetate 1:1) toafford the desired product 5 (1.16 g, 65%) as a colorless solid. ¹H NMR(400 MHz, CDCl₃): 1.16 (3H, t, J=7.2), 1.99-2.06 (1H, m), 2.25 (3H, s),2.28-2.36 (1H, m), 2.42-2.50 (1H, m), 2.55-2.64 (1H, m), 4.11 (1H, qd,J=7.1, 2.6), 4.62 (1H, dd, J=9.4, 2.7), 5.16 (1H, d, J=12.4), 4.25 (1H,d, J=12.4), 7.02-7.05 (2H, m), 7.35-7.39 (2H, m). ¹³C NMR (101 MHz,CDCl₃): 14.02, 21.08, 21.79, 30.99, 58.74, 61.82, 67.58, 121.75, 129.46,132.62, 150.70, 150.83, 169.28, 170.97, 172.93. Optical rotation: [α]²²_(D)−30.7° (c 0.298, CHCl3). IR (CHCl3): 2968 w, 2942 vw, 2876 vw, 1797s, 1753 vs, 1717 s, sh, 1609 w, 1597 vw, 1510 m, 1476 vw, 1463 w, 1450w, 1447 w, 1421 w, 1402 w, sh, 1380 m, 1372 m, 1303 s, 1288 s, 1259 m,1198 vs, 1166 s, 1107 vw, 1045 m, 1019 m, 1012 m, sh, 913 w, 846 w, 596w, cm⁻¹. ESI MS: 372 ([M+Na]⁺). HR ESI MS: calcd for C₁₇H₁₉O₇NNa372.10537; found 372.10541.

Ethyl 2-((((4-acetoxybenzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate(6)

Trimethylsilyl diazomethane solution (2M in diethyl ether, 1.7 mL, 3.43mmol, 1.2 equiv.) was dissolved in absolute THF (17 mL) under argon andcooled to −98° C. A solution of n-butyllithium (2.5 M in hexanes, 1.4mL, 3.52 mmol, 1.23 equiv.) was added dropwise and the solution wasstirred at −98° C. for 30 min. The resultant mixture was transferred viacannula to a solution of previous compound 5 (1.0 g, 2.86 mmol, 1equiv.) in absolute THF (27 mL) at −116° C. The reaction mixture wasslowly warmed to −78° C. and then quenched with saturated NH₄Cl. Then itwas extracted with ethyl acetate (3×50 mL), the combined organic layerswere washed with water (40 mL), brine (40 mL) and dried over anhydrousMgSO₄. The organic solvent was evaporated in vacuo. The residue waschromatographed on silica gel (chloroform—acetone 20:1) to afford thedesired product 6 (460 mg, 41%) as a yellowish solid. ¹H NMR (400 MHz,CDCl₃): 1.25 (3H, t, J=7.1), 1.92-2.03 (1H, m), 2.13-2.23 (1H, m), 2.28(3H, s), 2.32-2.43 (1H, m), 4.18 (1H, q, J=7.1), 4.29-4.34 (1H, m), 5.04(1H, d, J=12.4), 5.10 (1H, d, J=12.1) 5.22 (1H, s), 7.31 (1H, d, J=8.2),7.04-7.07 (2H, m), 7.34-7.37 (2H, m). ¹³C NMR (101 MHz, CDCl₃): 14.22,21.19, 27.53, 36.45, 53.62, 61.78, 66.38, 121.78, 129.52, 134.01,150.59, 156.01, 169.51, 171.90, 193.60. Optical rotation: [α]²²_(D)+15.5° (c 0.129, CHCl₃). IR (CHCl₃): 3428 w, 3116 w, 2966 w, 2110 s,1721 vs, 1742 s, sh, 1641 m, 1609 w, sh, 1595 vw, sh, 1509 s, 1418 vw,1381 s, sh, 1371 s, 1344 m, 1197 vs, 1166 m, 1106 vw, 1053 m, br, 1019m, 1012 m, sh, 913 w, 848 w, 595 w, 492 w cm⁻¹. ESI MS: 414 ([M+Na]⁺).HR ESI MS: calcd for C₁₈H₂₁O₇N₃Na 414.12717; found 414.12713.

Synthesis of 1-methylethyl2,6-bis[4-(1λ⁵-diazynylidene)-3-oxobutyl]-9-methyl-7-oxo-4-phenoxy-8-oxa-3,5-diaza-4-phosphadecan-1-oate4-oxide (7) 1-methylethyl2,6-bis[4-(1λ⁵-diazynylidene)-3-oxobutyl]-9-methyl-7-oxo-4-phenoxy-8-oxa-3,5-diaza-4-phosphadecan-1-oate4-oxide (7)

Referring to scheme 1, isopropyl DON 3 (100 mg, 0.38 mmol) was dissolvedin absolute dichloromethane (3 mL) and diisopropylethyl amine (327 μL,1.88 mmol, 4 equiv.) was added. This solution was cooled to 0° C. andphenyl dichlorophosphate (31.6 μL, 0.21 mmol, 0.45 equiv.) was addeddropwise. The reaction mixture was stirred at 0° C. for 30 min and thenthe cooling bath was removed. The reaction mixture was then stirred atroom temperature for 2 h. The organic solvent was evaporated in vacuo.The residue was chromatographed on silica gel (ethyl acetate—methanol40:1) to afford the desired product (78 mg, 66%) as a yellow oil. ¹H NMR(400 MHz, CDCl₃): 1.21-1.24 (12H, m), 1.81-1.93 (2H, m), 2.09-2.19 (2H,m), 2.24-2.52 (4H, m), 3.61-3.70 (2H, m), 3.90-3.98 (2H, m), 4.99 (1H,hept, J=6.3) 4.99 (1H, hept, J=6.2), 5.24 (1H, s), 5.33 (1H, s),7.10-7.14 (1H, m), 7.17-7.20 (2H, m), 7.28-7.31 (2H, m). ¹³C NMR (101MHz, CDCl₃): 21.81 (4C), 29.26, 29.43, 36.21 (2C), 53.78 (d,J_(C,P)=1.7), 53.97, 69.54, 69.66, 120.43 (d, J_(C,P)=4.8), 124.76,129.76 (2C), 151.03 (2C, d, J_(C,P)=6.8), 172.47 (d, J_(C,P)=5.5),172.64 (d, J_(C,P)=5.2), 194.07 (2C). ³¹P NMR (101 MHz, CDCl₃): 11.08.Optical rotation: [α]²² _(D)+6.4° (c 0.313, CHCl₃). IR (CHCl₃): 3099 w,3303 w, 2104 vs, 1732 s, 1639 s, 1592 w, 1492 m, 1385 sh, m, 1376 s,1240 sh, m, 1211 s, 1183 sh, m, 1167 m, 1144 m, 1132 sh, m, 1106 s, 1072w, 1025 w, 1006 m, 923 m, 832 sh, w, 771 w, m, 692 w cm⁻¹.

ESI MS: 587 ([M+Na]⁺). HR ESI MS: calcd for C₂₄H₃₃N₆O₈PNa 587.19897;found 587.19899.

Synthesis of isopropyl2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (9) Isopropyl2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-diazo-5-oxohexanoate(8)

Referring to scheme 1, Fmoc-NH-(L)-Leu-COOH (874 mg, 2.47 mmol, 1.1 eq.)and HBTU (1023 mg, 2.70 mmol, 1.2 eq.) were suspended in dry DCM (15mL). DIEA (872 mg, 1.18 mL, 6.75 mmol, 3 eq.) and then the solution ofNH₂-DON—COOiPr (448 mg, 2.25 mmol, 1 eq.) in dry DCM (5 mL) were addedby syringe. The reaction mixture was stirred for 2 h at room temperatureunder an inert atmosphere. DCM (20 mL) was added and the organic phasewas washed with sat. NaHCO₃ (40 mL), 1M HCl (40 mL), water (2×40 mL) andsat. NaCl (40 mL), dried over MgSO₄. DCM was evaporated. The crudeproduct was purified by column chromatography (DCM/EtOAc 4:1, R_(f)0.27) and light yellow solid (949 mg) was obtained in a 79% yield. ¹HNMR (400 MHz, CDCl₃): 0.91-0.96 (6H, m), 1.23-1.25 (6H, m), 1.51-1.73(3H, m), 1.93-2.02 (1H, m), 2.14-2.24 (1H, m), 2.26-2.44 (2H, m),4.16-4.24 (2H, m), 4.33-4.37 (1H, m), 4.39-4.44 (1H, m), 4.47-4.52 (1H,m), 4.96-5.08 (1H, m), 5.18 (1H, s), 5.36 (1H, d, J=8.2), 6.84 (1H, d,J=7.8), 7.30 (2H, tt, J=7.4, 1.2), 7.37-7.42 (2H, m), 7.58 (2H, d,J=7.4), 7.75 (2H, d, J=7.5). ¹³C NMR (101 MHz, CDCl₃): 21.70, 21.72,22.04, 22.92, 24.66, 27.04, 36.37, 41.75, 47.14, 52.05, 53.56, 67.08,69.47, 119.98, 120.01, 125.06, 125.13, 127.10, 127.11, 127.74, 127.75,141.28 (2C), 143.76, 143.81, 156.14, 170.95, 172.29, 193.80. Opticalrotation: [α]²² _(D)−6.1° (c 0.472, CHCl₃). IR (CHCl₃): 3304 m, sh, 3067w, 3018 w, 2105 s, 1730 s, 1704 s, 1659 vs, 1639 sh, m, 1612 sh, w, 1580sh, w, 1539 s, 1478 m, 1451 m, 1467 m, 1386 sh, s, 1375 s, 1244 s, 1172sh, m, 1145 m, 1106 s, 834 sh, w, 759 m, 740 s, 621 m, 427 cm⁻¹. ESI MS:571 ([M+Na]⁺). HR ESI MS: calcd for C₂₄H₃₃O₄N₄Na 571.25271; found571.25271.

Isopropyl 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (9)

The same method of preparation as for previous compound 3 was used.Referring to scheme 1, compound 8 (90 mg, 0.164 mmol), dichloromethane(1 mL), piperidine (41 μL 0.41 mmol, 2.5 equiv.). Chromatography onsilica gel (chloroform—methanol 20:1). Product (31 mg, 66%) as a yellowsolid. ¹H NMR (400 MHz, CDCl₃): 0.94 (3H, d, J=6.3), 0.98 (3H, d,J=6.4), 1.25 (3H, d, J=2.8), 1.26 (3H, d, J=2.8), 1.35-1.42 (1H, m),1.76-1.79 (2H, m), 1.95-2.04 (1H, m), 2.17-2.25 (1H, m), 2.33-2.49 (2H,m), 3.48 (1H, dd, J=9.7, 4.1), 4.52 (1H, td, J=8.5, 4.7), 5.04 (1H,hept, J=6.3), 5.35 (1H, s), 7.87 (1H, d, J=8.3). ¹³C NMR (101 MHz,CDCl₃): 27.59, 28.19, 33.40, 66.39, 80.90, 124.87, 128.33, 128.35,128.66, 136.10, 140.58, 166.01, 172.75. Optical rotation: [α]²² _(D)+°(c 0.33, CH₂Cl₂). IR (CHCl₃): 3412 w, 3343 w, 2110 vs, 1731 s, 1663 s,1643 sh, s, 1603 sh, w, 1510 s, 1413 w, 1386 sh, s, 1376 sh, s, 1370 sh,s, 1349 m, 1145 m, 1105 s cm⁻¹. ESI MS: 327 ([M+H]⁺). HR ESI MS: calcdfor C₁₅H₂₇O₄N₄Na 327.20268; found 327.20280.

Synthesis of 1-methylethylL-leucyl-L-leucyl-6-(1l⁵-diazynylidene)-5-oxo-L-norleucinate (11)1-MethylethylN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-leucyl-L-leucyl-6-(1l⁵-diazynylidene)-5-oxo-L-norleucinate(10)

Fmoc-NH-(L)-Leu-COOH (224 mg, 0.634 mmol, 1.1 equiv.) and 2.benzotriazol-1-yl-tetramethyluronium tetrafluoroborate (TBTU) (222 mg,0.69 mmol, 1.2 equiv.) were suspended in absolute dichloromethane (5 mL)and diisopropylethyl amine (301 μL, 1.73 mmol, 3 equiv.) was added. Thereaction mixture was stirred at room temperature for 30 min. and thenthe solution of 9 (188 mg, 0.58 mmol) in dry dichloromethane (3 mL) wereadded by syringe. The reaction mixture was stirred for 2 h at roomtemperature under inert atmosphere. The organic solvent was evaporatedin vacuo. The residue was chromatographed on silica gel (hexane:ethylacetate, 1:1) to afford the desired product 10 (188 mg, 49%) as ayellowish amorphous solid. ¹H NMR (400 MHz, CDCl₃): 0.86-0.98 (12H, m),1.24 (3H, d, J=5.0), 1.25 (3H, d, J=5.0), 1.47-1.56 (2H, m), 1.57-1.72(4H, m), 1.91-2.04 (1H, m), 2.14-2.24 (1H, m), 2.26-2.48 (2H, m),4.14-4.22 (1H, m), 4.21 (1H, t, J=6.8), 4.36-4.49 (4H, m), 5.03 (1H,hept, J=6.3), 5.14 (1H, d, J=7.5), 5.32 (1H, s), 6.41 (1H, d, J=7.7),6.83 (1H, d, J=6.8), 7.32 (2H, tt, J=7.4, 1.3), 7.41 (2H, tt, J=7.5,1.5), 7.58 (2H, d, J=7.5), 7.77 (2H, tt, J=7.6, 1.1). ¹³C NMR (101 MHz,CDCl₃): 21.82, 21.84, 21.96, 22.22, 22.91, 23.14, 24.83 (2C), 27.22,36.46, 41.29, 41.39, 47.26, 51.94, 52.12, 53.74, 55.00, 67.22, 69.54,120.12, 120.14, 125.11, 125.15, 127.22 (2C), 127.88, 127.89, 141.42(2C), 143.77, 143.89, 156.42, 171.03, 171.81, 172.39, 193.97. Opticalrotation: [α]²² _(D)−25.2° (c 0.385, CHCl₃). IR (CHCl3): 3426 m, 3332 w,sh, 3116 w, 3068 w, 2961 s, 2873 m, 2110 s, 1726 vs, 1672 vs, 1640 sh,m, 1610 sh, w, 1579 sh, w, 1541 m, 1506 vs, 1479 m, 1468 m, 1387 s, 1371sh, s, 1377 s, 1349 m, 1286 m, 1234 s, 1171 sh, m, 1146 m, 1105 s, 1046m, 1023 sh, w, 824 w, 585 w, 488 w, sh, 426 w cm⁻¹. ESI MS: 684([M+Na]⁺). HR ESI MS: calcd for C₃₆H₄₇O₇N₅Na 684.33677; found 684.33607.

1-MethylethylL-leucyl-L-leucyl-6-(1l⁵-diazynylidene)-5-oxo-L-norleucinate (11)

Compound 10 (180 mg, 0.272 mmol), was dissolved in dichloromethane (4mL). Piperidine (67 μL 0.68 mmol, 2.5 equiv.) was added and the reactionmixture was stirred at room temperature for 4 h. The organic solvent wasevaporated in vacuo. The residue was chromatographed on silica gel(chloroform:methanol, 30:1) to afford the desired product 11 (80 mg,67%) as a yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃): 0.88-0.98(12H, m), 1.24 (3H, d, J=5.2), 1.26 (3H, d, J=5.3), 1.37-1.47 (1H, m),1.50-1.79 (5H, m), 1.91-2.07 (1H, m), 2.08-2.28 (3H, m), 2.31-2.50 (2H,m), 3.52 (1H, dd J=9.5, 3.8), 4.39 (1H, td, J=8.7, 5.3), 4.46 (1H, td,J=8.0, 4.6), 5.03 (1H, hept, J=6.2), 5.37 (1H, s), 7.02 (1H, d, J=7.4),7.77 (1H, d, J=8.1). ¹³C NMR (101 MHz, CDCl₃): 21.46, 21.83, 21.86,22.05, 23.09, 23.53, 24.95, 25.01, 27.13, 36.52, 40.89, 43.94, 51.55,52.16, 53.61, 55.01, 69.49, 171.09, 172.36, 175.91, 194.21. Opticalrotation: [α]²² _(D)−31.7° (c 0.439, CHCl₃). IR (CHCl3): 3415 w, 3343 w,br, 3117 w, 2984 m, 2961 s, 2936 m, 2873 m, 2855 w, 2110 vs, 1731 s,1665 vs, br, 1653 vs, br, 1630 s, sh, 1509 s, 1468 m, 1454 w, 1450 w,1440 w, 1386 s, 1377 s, 1370 s, 1349 m, 1201 m, 1183 w, 1146 m, 1105 scm⁻¹. ESI MS: 440 ([M+H]⁺). HR ESI MS: calcd for C₂₁H₃₈O₅N₅ 440.28675;found 440.28674.

Synthesis of 1-methylethyl6-(1λ⁵-diazynylidene)-N-{[(5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy]carbonyl}-5-oxo-L-norleucinate(13) 2,5-Dioxopyrrolidin-1-yl-((5-methyl-2-oxo-1,3-dioxol-4-yl)methylcarbonate (12)

4-(Hydroxymethyl)-5-methyl-1,3-dioxol-2-one (1.00 g, 7.89 mmol, 1 eq.)was dissolved in dry Et₂O (30 ml) and the solution was cooled to 0° C.At this temperature pyridine (608 mg, 619 μL, 7.69 mmol, 1 eq.) wasadded and finally the solution of S-ethyl carbonochloridothionate (1.04g, 874 μL, 8.38 mmol, 1.09 eq.) in dry Et₂O (8 mL) was added bydropwise. The mixture was stirred for 1 h at 0° C. and overnight at rt(18 h). Et₂O was removed by vacuo and DCM (70 mL) was added. Thereaction mixture was washed with sat. NaHCO3 (40 mL) and with water(3×40 mL), dried with MgSO4 and DCM was removed by rotavap. The crudeproduct was purified by column chromatography (hexane/EtOAc 5:1, R_(f)0.24). A light yellow liquid (1.29 g) was obtained in 77% yield. S-EthylO-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl) carbonothioate (800 mg, 3.67mmol, 1 eq.) and N-hydroxysuccinimide (844 mg, 7.33 mmol, 2 eq.) weresuspended in dry DCM (8 mL). The solution was cooled to 0° C. andperacetic acid (836 mg (100%), 2.32 g (36%), 11.00 mmol, 3 eq., 36%solution in acetic acid) was added by dropwise in 10 minutes. The finalmixture was stirred for 30 minutes at 0° C. and 2 h at rt. DCM (20 mL)was added and the organic phase was washed with water (2×35 mL) and sat.NaCl (35 mL). DCM was evaporated and the product 12 was obtained as acolorless solid compound (750 mg) in 76% yield. ¹H NMR (400 MHz, CDCl₃):2.20 (3H, s), 2.86 (4H, s), 5.05 (2H, s). ¹³C NMR (101 MHz, CDCl₃):9.67, 25.58, 59.89, 131.61, 142.20, 151.65, 168.39. IR (CHCl3): 1842 m,1824 s, 1819 s, 1792 s, 1749 vs, 1431 w, 1386 w, 1309 m, 1195 s, 935 w,900 w, 811 vw cm⁻¹. ESI MS: 294 ([M+Na]⁺). HR ESI MS: calcd forC₁₀H₉O₈NNa 294.02204; found 294.02213.

1-Methylethyl6-(1λ⁵-diazynylidene)-N-{[(5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy]carbonyl}-5-oxo-L-norleucinate(13)

Referring to scheme 2, compound 12 (70 mg, 0.258 mmol, 1.1 equiv) wasdissolved in absolute dichloromethane (2 mL). This solution was cooledto 0° C. and a solution of 3 (50 mg, 0.235 mmol) in dichloromethane (1mL) was added dropwise. Reaction mixture was stirred 15 min at 0° C.cooling bath was removed. The reaction mixture was then stirred at roomtemperature for 1 h. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform:acetone, 10:1) toafford the desired product 13 (72 mg, 83%) as a yellow oil. ¹H NMR (400MHz, CDCl₃): 1.24 (3H, d, J=3.4), 1.26 (3H, d, J=5.6), 1.94-2.04 (1H,m), 2.13-2.24 (1H, m), 2.16 (3H, s), 2.32-2.49 (2H, m), 4.23-4.28 (1H,m), 4.77-4.87 (2H, m), 5.04 (1H, hept, J=6.3), 5.28 (3H, s), 5.66 (1H,d, J=7.9). ¹³C NMR (101 MHz, CDCl₃): 9.51, 21.82, 21.85, 27.32, 36.33,53.86, 54.57, 69.79, 133.93, 140.07, 152.31, 155.25, 171.12, 193.50.Optical rotation: [α]²² _(D)+15.1° (c 0.417, CHCl3). IR (CHCl3): 3424 w,3012 w, 2984 w, 2935 w, 2111 s, 1836 sh, s, 1821 vs, 1736 sh, vs, 1725vs, 1641 m, 1603 sh, w, 1509 s, 1391 sh, m, 1383 s, 1366 sh, m, 1105 scm⁻¹. ESI MS: 392 ([M+Na]⁺). HR ESI MS: calcd for C₁₅H₁₉O₈N₃Na392.10644; found 392.10650.

Synthesis of 1-methylethyl6-(1λ⁵-diazynylidene)-N-({1-[(2,2-dimethylpropanoyl)oxy] ethoxy}carbonyl)-5-oxo-L-norleucinate (14a) and (14b1-((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl pivalate (LTP 174)

LTP 174 was prepared according to the literature procedure (WO2008033572 A1).

1-Methylethyl 6-(1λ⁵-diazynylidene)-N-({1-[(2,2-dimethylpropanoyl)oxy]ethoxy} carbonyl)-5-oxo-L-norleucinate (14a) and (14b)

Referring to scheme 2, compound 14a and 14b were prepared from 3 (200mg, 0.938 mmol) as described for the preparation of 13 usingdichloromethane (8 mL) and1-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl pivalate (297 mg,1.032 mmol, 1.1 equiv.) to give isopropyl(2S)-6-diazo-5-oxo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoate14 (as a mixture of diastereoisomers), followed by chromatography onsilica gel (chloroform:acetone, 20:1) yielding two diastereoisomers:diastereoisomer #1 arbitrarily designated 14a (90 mg, R_(f)=0.25) anddiastereoisomer #2 arbitrarily designated 14b (50 mg, R_(f)=0.2) as ayellow oils (39%). ¹H NMR (400 MHz, CDCl₃): 1.18 (9H, s), 1.25 (3H, d,J=3.0), 1.26 (3H, d, J=3.1), 1.46 (3H, d, J=5.4), 1.94-2.04 (1H, m),2.15-2.24 (1H, m), 2.31-2.49 (2H, m), 4.24-4.30 (1H, m), 5.04 (1H, hept,J=6.2), 5.28 (1H, s), 5.50 (1H, d, J=7.9), 6.77 (1H, q, J=5.4). ¹³C NMR(101 MHz, CDCl₃): 19.78, 21.83, 21.85, 27.00 (3C), 27.79, 36.56, 38.79,53.58, 69.76, 89.70, 153.90, 171.16, 176.61, 193.59. IR (CHCl3): 3427 w,3116 w, 2984 m, 2960 sh, m, 2937 w, 2875 w, 2857 sh, w, 2110 s, 1745 sh,vs, 1730 sh, vs, 1641 m, 1508 s, 1480 m, 1467 sh, w, 1461 w, 1455 sh, w,1392 sh, s, 1383 sh, s, 1377 s, 1371 sh, s, 1365 sh, s, 1350 m, 1027 mcm⁻¹. Optical rotation (14a): [α]²² _(D)+22.0° (c 0.191, CHCl3). Opticalrotation (14b): [α]²² _(D)+7.6° (c 0.158, CHCl3). ESI MS (14a): 408([M+Na]⁺). ESI MS (14b): 408 ([M+Na]⁺). HR ESI MS (14a): calcd forC₁₇H₂₇O₇N₃Na 408.17412; found 408.17425. HR ESI MS (14b): calcd forC₁₇H₂₇O₇N₃Na 408.17412; found 408.17421. Diastereomer 14b was used inthe biological studies described herein. The absolute stereochemistry of14b has not been determined.

Synthesis of Isopropyl6-diazo-2-(((1-((3-methylbutanoyl)oxy)ethoxy)carbonyl)amino)-5-oxohexanoate(JAM0335) 1-((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethylisobutyrate (LTP 150)

LTP 150 was prepared according the literature procedure (Magill et al.,1957.

Isopropyl6-diazo-2-(((1-((3-methylbutanoyl)oxy)ethoxy)carbonyl)amino)-5-oxohexanoate(JAM0335)

The same method of preparation as for previous compound 13 was used.Referring to scheme 2, LTP150 (74 mg, 0.258 mmol, 1.1 equiv.),dichloromethane (2 mL). Compound 3 (50 mg, 0.235 mmol), dichloromethane(1 mL), Chromatography on silica gel (chloroform—acetone 20:1). Product(31 mg, 66%) was isolated as a yellow oil. ¹H NMR (400 MHz, CDCl₃): 1.48(9H, s), 2.54-2.64 (4H, m), 5.11 (2H, s), 5.48 (1H, m), 6.07 (1H, d,J=1.2), 7.30-7.40 (5H, m). ¹³C NMR (101 MHz, CDCl₃): 27.59, 28.19,33.40, 66.39, 80.90, 124.87, 128.33, 128.35, 128.66, 136.10, 140.58,166.01, 172.75. ESI MS: 313 ([M+Na]⁺). HR ESI MS: calcd for C₁₇H₂₂O₄Na313.14103; found 313.14103.

Synthesis of 1-methylethyl6-(1λ⁵-diazynylidene)-N-({1-[(2-methylpropanoyl)oxy]ethoxy}carbonyl)-5-oxo-L-norleucinate (15) 1-Methylethyl6-(1λ⁵-diazynylidene)-N-({1-[(2-methylpropanoyl)oxy]ethoxy}carbonyl)-5-oxo-L-norleucinate (15)

Compound 15 was prepared from 3 (300 mg, 1.4 mmol) as described for thepreparation of 13 using1-[[[(2,5-Dioxopyrrolidin-1-yl)oxy]carbonyl]oxy]ethyl 2-methylpropanoate(prepared by WO 2005066122, 423 mg, 1.55 mmol, 1.1 equiv.) anddichloromethane (4 mL), followed by chromatography on silica gel(chloroform:acetone, 20:1). A yellow oil (426 mg, 82%). ¹H NMR (400 MHz,CDCl₃): 1.13-1.17 (6H, m), 1.24-1.26 (6H, m), 1.46 (3H, d, J=5.4),1.89-2.03 (1H, m), 2.15-2.26 (1H, m), 2.31-2.58 (3H, m), 4.25-4.31 (1H,m), 5.03 (1H, hept, J=6.3), 5.29 and 5.34 (1H, 2×s), 5.48 and 5.52 (1H,2×d, J=8.2 and 7.9), 6.75-6.81 (1H, m). ¹³C NMR (101 MHz, CDCl₃): 18.78(2C), 19.83, 21.81, 21.84, 27.81, 33.98, 36.40, 53.47, 54.92, 69.75,89.53, 153.87, 171.12, 175.19, 193.60. IR (CHCl3): 3427 w, 3116 w, 2983m, 2940 w, 2879 w, 2110 s, 1746 vs, 1733 vs, 1641 m, 1509 s, 1449 m,1387 s, 1349 s, 1341 m, 1321 m, 1030 s cm⁻¹. ESI MS: 394 ([M+Na]⁺). HRESI MS: calcd for C₁₆H₂₅O₇N₃Na 394.15847; found 394.15859.

Synthesis of 4-acetoxybenzyl4-(4-diazo-3-oxobutyl)-5-oxooxazolidine-3-carboxylate (17)3-(3-(((4-acetoxybenzyl)oxy)carbonyl)-5-oxooxazolidin-4-yl)propanoicacid (16)

Referring to scheme 3, a solution of phosgene (15% vol.) (2.12 g, 2.25mL, 21.43 mmol, 2.21 eq.) in PhCH₃ (15 mL) was cooled to 0° C.4-(Hydroxymethyl)phenyl acetate (1.61 g, 9.70 mmol, 1 eq.) in PhCH₃ (15mL) was added dropwise over 15 min. The reaction mixture was stirred for1 h at 0° C. and then at room temperature overnight (20 h). Solvent wasevaporated and the crude product 4-chlorocarbonyl)oxy]methyl}phenylacetate (LTP 086) was used for the next step without purification.L-glutamic acid (951 mg, 6.46 mmol, 1 eq.) was suspended in water (8 mL)and NaHCO₃ (1.37 g, 16.30 mmol, 2.52 eq.) was added in few portions.After 15 min of stirring, crude LTP 086 (2.2 g, 9.70 mmol, 1 eq.) wasadded by syringe over 2 minutes at room temperature. The mixture wasstirred at room temperature overnight (20 h). 1 M HCl was added untilthe mixture was at a pH of 1 and the water phase was then extracted withEtOAc (10×15 mL). Combined organic fractions were washed with sat. NaCl(150 mL), dried over MgSO₄ and the solvent was evaporated. The crudeproduct N-[[[4-(acetyloxy)phenyl]methoxy]carbonyl]-L-gluamamic acid (LTP087) (2.2 g, 6.48 mmol, 1 eq.) was used in the next step withoutpurification. LTP 087 (2.2 g, 6.48 mmol, 1 eq.) was dissolved in PhCH₃(45 mL). Paraformaldehyde (389 mg, 12.97 mmol, 2 eq.) andp-Toluenesulfonic acid (PTSA) (123 mg, 0.648 mmol, 0.1 eq.) were addedand the mixture was refluxed for 1 h. Toluene was evaporated and thecrude product was purified by column chromatography (CHCl₃/MeOH 20:1).The desired product 16 was obtained as a colorless viscous oil (280 mg)in a 12% yield over three steps.

4-acetoxybenzyl 4-(4-diazo-3-oxobutyl)-5-oxooxazolidine-3-carboxylate(17)

The compound LTP088 (257 mg, 0.732 mmol) was dissolved in absolute THF(3 mL), cooled to −15° C. and triethylamine (153 μL, 1.097 mmol, 1.5equiv.) was added dropwise. Then ethylchloroformate was added and thereaction mixture was stirred at −15° C. for 1.5 h. Then a solution ofdiazomethane was added and the reaction mixture was stirred for another30 min at −15° C. and then the cooling bath was removed. The reactionmixture was then stirred at room temperature for 2 h. The organicsolvent was evaporated in vacuo. The residue was chromatographed onsilica gel (hexane—ethyl acetate 1:1) to afford the desired product (220mg, 66%) as a yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃): 2.14 (2H,m), 2.29 (3H, s), 2.32-2.56 (2H, m), 4.34 (1H, t, J=6.2), 5.11-5.25 (4H,m), 5.51 (1H, s), 7.07-7.10 (2H, m), 7.37-7.40 (2H, m). ¹³C NMR (101MHz, CDCl₃): 21.19, 25.80, 35.36, 54.12, 67.49, 77.89, 122.06 (2C),129.96 (2C), 130.02, 150.97, 152.98, 169.49, 171.81, 192.64. IR (CHCl₃):3116 w, 2964 vw, 2922 w, 2111 s, 1802 s, 1768 m, sh, 1756 s, 1716 vs,1642 m, 1610 w, 1597 vw, sh, 1510 m, 1423 m, 1410 s, 1383 m, 1371 s,1355 s, 1197 vs, 1167 s, 1128 m, 1106 w, 1019 m, 1013 m, 914 m, 850 w,596 w, 492 w, cm⁻¹. ESI MS: 398 ([M+Na]⁺). HR ESI MS: calcd forC₁₇H₁₇O₇N₃Na 398.09587; found 398.09596.

Synthesis of ethyl 2-(2-aminopropanamido)-6-diazo-5-oxohexanoate (22)1-(9H-fluoren-9-ylmethyl)-2-ethyl-5-oxopyrrolidine-1,2-dicarboxylate(18)

Referring to scheme 4, 5-Oxo-L-proline ethyl ester (4.00 g, 25.45 mmol,1 eq.) was dissolved in absolute THF (120 mL) under inert and cooled to−78° C. A solution of LiHMDS (1M in THF, 24.2 mL, 24.18 mmol, 0.95 eq.)was added dropwise and the solution was stirred at the same temperaturefor 20 min. The resultant yellow mixture was transferred via cannula toa solution of Fmoc chloride (32.9 g, 127.3 mmol, 5 eq.) in absolute THF(90 mL) at −78° C. The reaction mixture was stirred at −78° C. for 2 hand at rt overnight (18 h). After this period the reaction was quenchedwith saturated NH₄Cl (34 mL) and water (18 mL). Water phase wasextracted with EtOAc (60 mL) and combined organic parts were washed withbrine (2×100 mL) and dried over anhydrous MgSO₄. The organic solvent wasevaporated under vacuo. The residue was chromatographed on silica gel(hexane:EtOAc 3:1 to 1:1) and finally on reverse LC (MeOH:H₂O, 2:1 to100% MeOH) to afford the desired product 18 (8.40 g, 87%) as acolourless solid. ¹H NMR (400 MHz, CDCl₃): 1.26 (3H, d, J=7.1), 2.12(1H, ddt, J=13.4, 9.4, 2.9), 2.40 (1H, ddt, J=13.4, 10.8, 9.3), 2.57(1H, ddd, J=17.5, 9.2, 3.1), 2.72 (1H, ddd, J=17.5, 10.7, 9.4), 4.20(2H, q, J=7.1), 4.30 (1H, t, J=7.3), 4.44 (1H, dd, J=10.5, 7.4), 4.57(1H, dd, J=10.6, 7.3), 4.65 (1H, dd, J=9.4, 2.5), 7.33 (2H, tt, J=7.4,1.1), 7.41 (2H, tdd, J=6.9, 1.3, 0.6), 7.71 (1H, dd, J=7.5, 1.0), 7.75(1H, dd, J=7.5, 1.0), 7.77 (2H, dd, J=7.8, 1.0). ¹³C NMR (101 MHz,CDCl₃): 14.14, 21.96, 31.21, 46.62, 58.76, 61.92, 69.03, 119.99, 120.01,125.30, 125.44, 127.26 (2C), 127.91 (2C), 141.27, 141.29, 143.35,143.40, 151.44, 170.80, 172.99. Optical rotation: [α]²² _(D)−17.2° (c0.285, CHCl3). IR (CHCl3): 3068 m, 2985 m, 2941 w, 2898 w, 2875 w, 1797vs, 1745 vs, br, 1723 vs, 1609 w, 1580 vw, 1478 m, 1463 m, 1452 s, 1400m, sh, 1385 s, 1197 vs, 1116 vw, sh, 1104 m, 1097 w, sh, 1033 s, 621 m,426 w, cm⁻¹. ESI MS: 402 ([M+Na]⁺). HR ESI MS: calcd for C₂₂H₂₁O₅NNa402.1312; found 402.1313.

Ethyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoate(19)

Trimethylsilyl diazomethane solution (2M in diethyl ether, 7.9 mL, 15.81mmol, 1.2 eq.) was dissolved in absolute THF (70 mL) under argon andcooled to −98° C. A solution of n-butyllithium (2.5 M in hexanes, 6.5mL, 16.21 mmol, 1.23 eq.) was added dropwise and the solution wasstirred at −98° C. for 30 min. The resultant mixture was transferred viacannula to a solution of 18 (5.00 g, 13.18 mmol, 1 eq.) in absolute THF(120 mL) at −116° C. The reaction mixture was stirred for 30 min at thistemperature and then slowly warmed to −78° C. and quenched withsaturated NH₄Cl (150 mL). Water phase was extracted with ethyl acetate(3×50 mL), the combined organic layers were washed with brine (200 mL)and dried over anhydrous MgSO₄. The organic solvent was evaporated invacuo. The residue was chromatographed on silica gel (CHCl₃:acetone,20:) to afford the desired product 19 (4.42 g, 80%) as a yellowishsolid. ¹H NMR (400 MHz, CDCl₃): 1.29 (3H, t, J=7.1), 1.93-2.10 (1H, m),2.17-2.29 (1H, m), 2.33-2.53 (2H, m), 4.22 (3H, t, J=7.1), 4.32-4.43(3H, m), 5.27 (1H, bs), 5.56 (1H, d, J=8.1), 7.32 (2H, tt, J=7.4, 1.3),7.41 (2H, t, J=7.5), 7.60 (2H, t, J=6.6), 7.77 (2H, dd, J=7.6, 1.0). ¹³CNMR (101 MHz, CDCl₃): 14.30, 27.63, 36.56, 47.26, 53.63, 54.96, 61.89,67.17, 120.11, 120.14, 125.22, 125.24, 127.20 (2C), 127.86 (2C), 141.40,141.43, 143.78, 143.99, 156.18, 172.03, 193.65. Optical rotation: [α]²²_(D)+10.6° (c 0.265, CHCl3). IR (CHCl3): 3428 w, 3116 w, 3068 w, 2985 w,2942 w, 2907 vw, 2110 s, 1740 s, sh, 1721 vs, 1642 m, 1510 s, 1478 w,1465 w, 1451 m, 1381 s, 1105 w, 1052 m, 1033 m, 622 w, 426 w cm⁻¹. ESIMS: 444 ([M+Na]⁺). HR ESI MS: calcd for C₂₃H₂₃O₅N₃Na 444.15299; found444.15292.

Ethyl 2-amino-6-diazo-5-oxohexanoate (20)

Compound 20 was prepared from 19 (100 mg, 0.237 mmol) as described forthe preparation of 3 using dichloromethane (10 mL) and piperidine (58 μL0.59 mmol, 2.5 equiv), followed by chromatography on silica gel(chloroform:methanol, 30:1). A yellow oil (31 mg, 66%). ¹H NMR (400 MHz,CDCl₃): 1.27 (3H, t, J=7.1), 1.55 (2H, bs), 1.78-1.88 (1H, m), 2.06-2.17(1H, m), 2.40-2.54 (2H, bm), 3.44 (1H, dd, J=8.3, 5.1), 4.17 (2H, q,J=7.1), 5.27 (1H, s). ¹³C NMR (101 MHz, CDCl₃): 14.24, 29.56, 36.86,53.77, 54.56, 61.05, 175.58, 194.15. Optical rotation: [α]²² _(D)+° (c0.33, CH₂Cl₂). IR (CHCl3): 3410 w, vbr, 3327, vw, vbr, 2986 m, 2941 w,2910 w, 2874 w, 2110 s, 1739 vs, 1641 m, 1605 m, sh, 1586 m, 1552 w,1513 w, br, 1476 w, 1463 m, 1446 m, 1395 m, sh, 1377 s, 1200 s, 1115 m,1096 m cm⁻¹. ESI MS: 182 ([M−H₂O+H]⁺). HR ESI MS: calcd for C₁₂H₁₂N₃O₂182.0930; found 182.0931.

Ethyl2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-6-diazo-5-oxohexanoate(21)

Fmoc-NH-(L)-Ala-COOH (483 mg, 1.55 mmol, 1.1 eq.) and HBTU (642 mg, 1.69mmol, 1.2 eq.) were suspended in dry DCM (10 mL). DIEA (547 mg, 737 μL,4.32 mmol, 3 eq.) and then the solution of 20 (281 mg, 1.41 mmol, 1 eq.)in dry DCM (3 mL) were added by syringe. The reaction mixture wasstirred for 2 h at rt under inert atmosphere. DCM (15 mL) was added andthe organic phase was washed with sat. NaHCO₃ (25 mL), 1M HCl (25 mL),water (2×25 mL) and sat. NaCl (25 mL), dried oved MgSO₄. DCM wasevaporated. The crude product was purified by column chromatography(DCM:EtOAc, 1:1, R_(f) 0.40) to give 21 as a light yellow solid (452 mg,65%). ¹H NMR (400 MHz, CDCl₃): 1.28 (3H, t, J=7.5), 1.42 (3H, d, J=7.1),1.98-2.08 (1H, m), 2.16-2.28 (1H, m), 2.30-2.48 (2H, m), 4.15-4.30 (4H,m), 4.39 (2H, d, J=7.3), 4.53 (1H, td, J=8.0, 4.5), 5.21 (1H, s), 5.38(1H, d, J=7.4), 6.81 (1H, d, J=7.8), 7.32 (2H, t, J=7.5), 7.41 (2H, t,J=7.5), 7.60 (2H, d, J=7.5), 7.77 (2H, d, J=7.5). ¹³C NMR (101 MHz,CDCl₃): 14.25, 18.86, 27.00, 36.48, 38.74, 47.22, 50.65, 55.11, 61.84,67.24, 120.11, 120.13, 125.20, 125.24, 127.22 (2C), 127.87 (2C), 141.40(2C), 143.92 (2C), 156.01, 171.58, 172.47, 193.99. Optical rotation:[α]²² _(D)+0.4° (c 0.225, CHCl3). IR (CHCl3): 3424 m, 3330 w, br, 3116vw, 3068 vw, 2986 m, 2941 w, 2908 w, 2875 vw, 2110 s, 1731 vs, 1720 vs,sh, 1682 vs, 1639 m, 1585 vw, 1503 vs, 1478 m, 1451 s, 1377 s, 1233 s,1116 m, 1105 m, sh, 1095 w, 1032 m, 622 w, 424 w, cm⁻¹. ESI MS: 515([M+Na]⁺). HR ESI MS: calcd for C₂₆H₂₈O₆N₄Na 515.19011; found 515.19044.

Ethyl 2-(2-aminopropanamido)-6-diazo-5-oxohexanoate (22)

Compound 21 (225 mg, 0.457 mmol, 1 eq.) was dissolved in dry DCM (4 mL).Diethylamine (167 mg, 236 μL, 2.28 mmol, 5 eq.) was added by syringe.The reaction mixture was stirred for 6 h at rt under inert atmosphere.DCM was evaporated. The crude product was purified by columnchromatography (CHCl₃:MeOH, 15:1, R_(f) 0.07) to give 22 as a lightyellow solid (101 mg, 82%). ¹H NMR (400 MHz, CDCl₃): 1.22 (3H, t,J=7.1), 1.28 (3H, d, J=7.0), 1.89-1.94 (2H, m), 1.94-2.01 (1H, m),2.10-2.21 (1H, m), 2.27-2.43 (2H, m), 3.47 (1H, q, J=7.0), 4.13 (2H, q,J=7.1), 4.47 (1H, td, J=8.4, 4.9), 5.32 (1H, s), 7.81 (1H, d, J=8.4).¹³C NMR (101 MHz, CDCl₃): 14.28, 18.56, 27.44, 36.80, 51.63, 55.05,58.55, 61.74, 171.90, 175.25, 193.93. Optical rotation: [α]²² _(D)−31.1°(c 0.260, CHCl3+DMF). IR (CHCl3): 3393 w, 3336 vw, br, 3211 vw, br, 3116vw, 2958 m, 2927 s, 2871 m, 2856 m, 2110 s, 1736 m, 1684 vs, 1639 m,1517 w, 1379 s, 1115 vw, sh, 1097 w, cm⁻¹. ESI MS: 271 ([M+H]⁺). HR ESIMS: calcd for C₁₁H₁₉O₄N₄ 271.14008; found 271.14024.

Synthesis of 2-(2-aminopropanamido)-6-diazo-5-oxohexanoic acid (23)2-(2-Aminopropanamido)-6-diazo-5-oxohexanoic acid (23)

Referring to scheme 4, compound 22 (225 mg, 0.457 mmol, 1 eq.) wassuspended in EtOH (3 mL) and THF (3 mL). 1M solution of NaOH (16 mg, 397μL, 0.95 eq.) was added and the mixture was stirred for 15 minutes. Thereaction was quenched with 1 M formic acid (397 μL, 0.95 eq.) and after10 minutes of stirring the mixture was evaporated to dryness. The crudeproduct was purified by preparative HPLC with Et₃N/CH₃COOH buffer. Thedesired product 23 was obtained as a light orange solid (50 mg) in 52%yield. ¹H NMR (400 MHz, CDCl₃): 1.45 (3H, d, J=7.0), 2.06-2.22 (2H, m),2.45-2.56 (2H, m), 4.01-4.13 (2H, m), 5.86 (1H, brs).

Synthesis of ethyl2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (25) Ethyl2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-diazo-5-oxohexanoate(24)

Referring to scheme 4, Fmoc-NH-(L)-Leu-COOH (874 mg, 2.47 mmol, 1.1 eq.)and HBTU (1023 mg, 2.70 mmol, 1.2 eq.) were suspended in dry DCM (15mL). DIEA (872 mg, 1.18 mL, 6.75 mmol, 3 eq.) and then the solution of20 (448 mg, 2.25 mmol, 1 eq.) in dry DCM (5 mL) were added by syringe.The reaction mixture was stirred for 2 h at rt under inert atmosphere.DCM (20 mL) was added and the organic phase was washed with sat. NaHCO₃(40 mL), 1M HCl (40 mL), water (2×40 mL) and sat. NaCl (40 mL), driedoved MgSO₄. DCM was evaporated. The crude product was purified by columnchromatography (DCM:EtOAc, 4:1, R_(f) 0.27) to give 24 as a light yellowsolid (949 mg, 79%). ¹H NMR (400 MHz, CDCl₃): 0.95 (6H, d, J=5.9), 1.26(3H, t, J=7.5), 1.50-1.59 (1H, m), 1.62-1.75 (2H, m), 1.95-2.05 (1H, m),2.17-2.27 (1H, m), 2.29-2.45 (2H, m), 4.14-4.25 (4H, m), 4.34-4.45 (2H,m), 4.49-4.58 (1H, m), 5.19 (1H, s), 5.27 (1H, d, J=8.4), 6.80 (1H, d,J=7.6), 7.31 (2H, t, J=7.4), 7.40 (2H, t, J=7.8), 7.59 (2H, d, J=7.5),7.76 (2H, d, J=7.6). ¹³C NMR (101 MHz, CDCl₃): 14.12, 22.03, 22.92,24.66, 26.96, 36.33, 41.65, 47.14, 51.94, 53.56, 55.00, 61.73, 67.09,120.00, 120.03, 125.05, 125.13, 127.12 (2C), 127.76, 127.77, 141.29,143.64, 143.75, 143.79, 156.15, 171.46, 172.26, 193.81. Opticalrotation: [α]²² _(D)−9.7° (c 0.109, DMF). IR (CHCl3): 3304 m, sh, 3067w, 3018 w, 2105 s, 1730 s, 1704 s, 1659 vs, 1639 sh, m, 1612 sh, w, 1580sh, w, 1539 s, 1478 m, 1451 m, 1467 m, 1386 sh, s, 1375 s, 1244 s, 1172sh, m, 1145 m, 1106 s, 834 sh, w, 759 m, 740 s, 621 m, 427 cm⁻¹. ESI MS:557 ([M+Na]⁺). HR ESI MS: calcd for C₂₉H₃₄O₆N₄Na 557.23706; found557.23707.

Ethyl 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (25)

Referring to scheme 4, compound 24 (945 mg, 1.77 mmol, 1 eq.) wasdissolved in dry DCM (9 mL). Diethylamine (646 mg, 914 μL, 8.84 mmol, 5eq.) was added by syringe. The reaction mixture was stirred for 4 h atrt under inert atmosphere. DCM was evaporated. The crude product waspurified by column chromatography (CHCl₃:MeOH, 15:1, R_(f) 0.38) to give25 as a colorless solid (500 mg, 91%). ¹H NMR (400 MHz, CDCl₃): 0.94(6H, dd, J=14.0, 6.3), 1.27 (3H, t, J=7.1), 1.28-1.37 (1H, m), 1.45 (2H,s), 1.57-1.83 (2H, m), 1.92-2.08 (1H, m), 2.16-2.26 (1H, m), 2.26-2.49(2H, m), 3.39 (1H, dd, J=10.0, 3.9), 4.19 (2H, dq, J=7.1, 1.4), 4.54(1H, dt, J=8.5, 4.8), 5.30 (1H, s), 7.85 (1H, d, J=8.3). ¹³C NMR (101MHz, CDCl₃): 14.25, 21.40, 23.54, 24.97, 27.71, 36.82, 44.29, 51.48,53.60, 54.86, 61.65, 171.99, 175.99, 193.67. Optical rotation: [α]²²_(D)−54.8° (c 0.323, CHCl3). IR (CHCl3): 3412 w, 3343 w, 2110 vs, 1731s, 1663 s, 1643 sh, s, 1603 sh, w, 1510 s, 1413 w, 1386 sh, s, 1376 sh,s, 1370 sh, s, 1349 m, 1145 m, 1105 s cm⁻¹. ESI MS: 335 ([M+Na]⁺). HRESI MS: calcd for C₁₄H₂₅O₄N₄ 313.18703; found 313.18712.

Synthesis of 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoic acidtriethylammonium salt (26)2-(2-Amino-4-methylpentanamido)-6-diazo-5-oxohexanoic acidtriethylammonium salt (26)

Compound 25 (134 mg, 0.429 mmol, 1 eq.) was suspended in EtOH (3 mL) andTHF (3 mL). 1M solution of NaOH (16 mg, 408 μL, 0.95 eq.) was added andthe mixture was stirred for 15 minutes. The reaction was quenched with 1M formic acid (408 μL, 0.95 eq.) and after 10 minutes of stirring themixture was evaporated to dryness. The crude product was purified bypreparative HPLC with Et₃N/CH₃COOH buffer. The desired product 26 wasobtained as a light orange solid (46 mg) in 40% yield. ¹H NMR (400 MHz,CDCl₃): 1.10 (6H, d, J=6.4), 1.50-1.61 (1H, m), 1.63-1.83 (2H, m),1.96-2.06 (1H, m), 2.11-2.23 (1H, m), 2.33-2.48 (1H, m), 3.67 (1H, dd,J=8.6, 5.6), 4.27 (1H, dd, J=7.0, 5.1), 5.51 (1H, brs).

Synthesis of ethyl2-(2-(2-amino-4-methylpentanamido)-4-methylpentanamido)-6-diazo-5-oxohexanoate(28) Ethyl11-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5,8-diisobutyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecan-12-oate(27)

Referring to scheme 4, Fmoc-NH-(L)-Leu-COOH (125 mg, 0.352 mmol, 1.1eq.) and HBTU (146 mg, 0.384 mmol, 1.2 eq.) were suspended in dry DCM (3mL). DIEA (124 mg, 167 μL, 0.960 mmol, 3 eq.) and then the solution of25 (100 mg, 0.320 mmol, 1 eq.) in dry DCM (3 mL) were added by syringe.The reaction mixture was stirred for 3 h at rt under inert atmosphere.DCM (10 mL) was added and the organic phase was washed with sat. NaHCO₃(20 mL), 1M HCl (20 mL), water (2×20 mL) and sat. NaCl (20 mL), driedoved MgSO₄. DCM was evaporated. The crude product was purified by columnchromatography (DCM:EtOAc, 2:1, R_(f) 0.35) to give 27 as a colorlesssolid (145 mg, 79%). ¹H NMR (400 MHz, CDCl₃): 0.92 (6H, d, J=6.5), 0.95(6H, d, J=6.1), 1.28 (3H, t, J=7.1), 1.49-1.59 (2H, m), 1.60-1.74 (4H,m), 1.95-2.06 (1H, m), 2.18-2.28 (1H, m), 2.30-2.50 (2H, m), 4.17-4.27(4H, m), 4.32-4.64 (4H, m), 5.29 (1H, d, J=8.0), 5.35 (1H, s), 6.54 (1H,d, J=8.0), 7.00 (1H, d, J=7.7), 7.31 (2H, t, J=7.5, 2.6), 7.40 (2H, td,J=7.3, 2.2), 7.57 (2H, d, J=7.5), 7.76 (2H, d, J=7.5). ¹³C NMR (101 MHz,CDCl₃): 14.26, 21.96, 22.17, 22.94, 23.15, 24.85, 27.12, 29.84, 36.48,38.28, 41.17, 41.33, 47.27, 51.94, 52.03, 61.79, 67.22, 120.14, 120.16,125.10, 125.14, 127.23 (2C), 127.90, 127.91, 141.43, 143.75, 143.89,156.43, 171.54, 171.81, 172.39, 194.00. Optical rotation: [α]²²_(D)−34.5° (c 0.109, DMF). IR (CHCl3): 3426 m, 3317 w, br, 3116 w, 3068w, 2961 s, 2873 m, 2109 s, 1795 w, 1731 vs, 1719 vs, 1667 vs, br, 1635s, sh, 1508 vs, 1478 m, 1468 m, 1451 s, 1385 s, 1375 s, 1371 s, 1233 s,1045 m, 1033 m, 622 w, 426 w, cm⁻¹. ESI MS: 670 ([M+Na]⁺). HR ESI MS:calcd for C₃₅H₄₅O₇N₅Na 670.32112; found 670.32122.

Ethyl2-(2-(2-amino-4-methylpentanamido)-4-methylpentanamido)-6-diazo-5-oxohexanoate(28)

Compound 27 (136 mg, 0.210 mmol, 1 eq.) was dissolved in dry DCM (3 mL).Diethylamine (77 mg, 109 μL, 1.05 mmol, 5 eq.) was added by syringe. Thereaction mixture was stirred for 8 h at rt under inert atmosphere. DCMwas evaporated. The crude product was purified by column chromatography(CHCl₃/MeOH 20:1, R_(f) 0.30) to give 28 as a light yellow oil (77 mg,87%). ¹H NMR (400 MHz, CDCl₃): 0.92 (6H, t, J=6.0), 0.95 (6H, t, J=5.9),1.26 (3H, t, J=7.1), 1.34-1.41 (1H, m), 1.52-1.61 (1H, m), 1.60-1.79(4H, m), 1.95-2.03 (1H, m), 2.15-2.25 (1H, m), 2.30-2.52 (2H, m), 3.48(1H, dd, J=9.5, 4.1), 3.96 (2H, s), 4.18 (2H, q, J=7.1), 4.41 (1H, td,J=8.8, 5.4), 4.49 (1H, td, J=8.0, 4.6), 5.36 (1H, s), 7.08-7.17 (1H, m),7.74 (1H, d, J=8.2). ¹³C NMR (101 MHz, CDCl₃): 14.25, 21.51, 22.02,23.06, 23.45, 24.91, 24.97, 27.03, 36.49, 40.77, 43.77, 51.63, 52.04,53.54, 55.13, 61.76, 171.60, 172.44, 175.74, 194.29. Optical rotation:[α]²² _(D)−25.8° (c 0.124, DMF). IR (KBr): 3413 s, vbr, 3314 s, vbr,3072 m, br, 2105 s, 1739 s, 1655 vs, br, 1539 s, br, 1468 s, 1386 s,1370 s, 1208 s, br, 1029 s, cm⁻¹. ESI MS: 426 ([M+H]⁺). HR ESI MS: calcdfor C₂₀H₃₆O₅N₅ 426.27110; found 426.27124.

Synthesis of6-diazo-2-(((1-(isobutyryloxy)ethoxy)carbonyl)amino)-5-oxohexanoic acidtriethylammonium salt (29)6-Diazo-2-(((1-(isobutyryloxy)ethoxy)carbonyl)amino)-5-oxohexanoic acidtriethylammonium salt (29)

Referring to scheme 5, compound 20 (121 mg, 0.607 mmol, 1 eq.) wasdissolved in THF (5 mL). 1M solution of NaOH (24 mg, 607 μL, 1 eq.) wasadded and the mixture was stirred for 1 h. Water (2 mL) and1-[[[(2,5-Dioxopyrrolidin-1-yl)oxy]carbonyl]oxy]ethyl 2-methylpropanoate(LTP 150) (183 mg, 0.668 mmol, 1.1 eq.) in THF (3 mL) were added. Thesolution was stirred for next 2 h at rt. The reaction was quenched with1 M formic acid (607 μL, 1 eq.) and after 5 minutes of stirring themixture was evaporated to dryness. The crude product was purified bypreparative HPLC with Et₃N/CH₃COOH buffer to give 29 as a light orangesolid (106 mg, 53%). ¹H NMR (400 MHz, CDCl₃): 1.11 (6H, d, J=6.4),1.35-1.46 (3H, m), 1.84-2.32 (2H, m), 2.36-2.75 (2H, m), 4.15-4.48 (1H,m), 4.60-4.97 (1H, m), 5.70-6.12 (1H, m), 6.67-6.82 (1H, m), 8.40 (1H,bs).

Synthesis of6-diazo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)-5-oxohexanoic acidtriethylammonium salt (30)6-Diazo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)-5-oxohexanoic acidtriethylammonium salt (30)

Referring to scheme 5, compound 20 (89 mg, 0.446 mmol, 1 eq.) wasdissolved in THF (3 mL). 1M solution of NaOH (18 mg, 446 μL, 1 eq.) wasadded and the mixture was stirred for 1 h. Water (1 mL) and1-((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl pivalate (LTP174)(141 mg, 0.490 mmol, 1.1 eq.) in THF (1.5 mL) were added. The solutionwas stirred for next 2 h at rt. The reaction was quenched with 1 Mformic acid (446 μL, 1 eq.) and after 5 minutes of stirring the mixturewas evaporated to dryness. The crude product was purified by preparativeHPLC with Et₃N/CH₃COOH buffer to give 30 as a light orange solid (64 mg)in 42% yield. ¹H NMR (400 MHz, CDCl₃): 1.18 (9H, s), 1.36-1.59 (3H, m),1.94-2.36 (2H, m), 2.40-2.77 (2H, m), 4.18-4.48 (1H, m), 4.53-4.97 (1H,m), 5.45-6.07 (1H, m), 6.68-6.84 (1H, m), 8.87 (1H, bs).

Synthesis of ethyl6-diazo-2-(((1-(isobutyryloxy)ethoxy)carbonyl)amino)-5-oxohexanoate (31)Ethyl6-diazo-2-(((1-(isobutyryloxy)ethoxy)carbonyl)amino)-5-oxohexanoate (31)

1-[[[(2,5-Dioxopyrrolidin-1-yl)oxy]carbonyl]oxy]ethyl 2-methylpropanoate(226 mg, 0.828 mmol, 1.1 eq) was suspended in dry DCM (6 mL). Thereaction mixture was cooled to 0° C. and compound 20 (150 mg, 0.753mmol, 1 eq.) in dry DCM (3 mL) was added by drop wise. The mixture wasstirred for 15 minutes at 0° C. and then 1 h at rt. The crude productwas purified by column chromatography (EtOAc/hexane 1:10 to 1:2) and thedesired compound 31 was obtained in 56% yield (150 mg) as a yellow oil(mixture of two stereoisomers 1:1). ¹H NMR (400 MHz, CDCl₃, Stereoisomer1): 1.14 (6H, d, J=6.7), 1.27 (3H, t, J=7.1), 1.46 (3H, d, J=5.4),1.88-2.09 (1H, m), 2.16-2.29 (1H, m), 2.34-2.47 (2H, m), 2.51 (1H, sep,J=7.1), 4.19 (2H, q, J=7.1), 4.27-4.36 (1H, m), 5.29 (1H, brs), 5.49(1H, d, J=8.3), 6.78 (1H, q, J=7.7). ¹³C NMR (101 MHz, CDCl₃,Stereoisomer 1): 14.26, 18.78, 18.80, 19.84, 27.73, 33.99, 36.35, 53.39,54.92, 61.92, 89.54, 153.87, 171.62, 175.18, 193.54. ¹H NMR (400 MHz,CDCl₃, Stereoisomer 2): 1.16 (6H, d, J=6.7), 1.27 (3H, t, J=7.1), 1.46(3H, d, J=5.4), 1.88-2.09 (1H, m), 2.16-2.29 (1H, m), 2.34-2.47 (2H, m),2.51 (1H, sep, J=7.1), 4.20 (2H, q, J=7.1), 4.27-4.36 (1H, m), 5.29 (1H,brs), 5.53 (1H, d, J=8.1), 6.80 (1H, q, J=7.7). ¹³C NMR (101 MHz, CDCl₃,Stereoisomer 2): 14.26, 18.80, 18.85, 19.87, 27.84, 33.99, 36.47, 53.50,54.97, 61.92, 89.72, 153.95, 171.79, 175.30, 193.77. Optical rotation:[α]²² _(D)+19.8° (c 0.177, CHCl3). IR (CHCl3): 3428 w, 3116 w, 2981 m,2940 w, 2877 w, 2856 w, 2110 vs, 1741 vs, br, 1641 m, 1510 s, 1469 m,1388 s, 1377 s, 1232 m, 1199 m, cm⁻¹. ESI MS: 380 ([M+Na]⁺). HR ESI MS:calcd for C₁₅H₂₄O₇N₃ 358.16088; found 358.16118.

Synthesis of ethyl6-diazo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)-5-oxohexanoate (32)Ethyl 6-diazo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)-5-oxohexanoate(32)

Referring to scheme 6,1-((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl pivalate (238 mg,0.828 mmol, 1.1 eq) was suspended in dry DCM (6 mL). The reactionmixture was cooled to 0° C. and compound 20 (150 mg, 0.753 mmol, 1 eq.)in dry DCM (3 mL) was added by drop wise. The mixture was stirred for 15minutes at 0° C. and then 1 h at rt. The crude product was purified bycolumn chromatography (EtOAc/hexane 1:10 to 1:2) and the desiredcompound 32 was obtained in 68% yield (190 mg) as a yellow oil (mixtureof two stereoisomers 1:1). ¹H NMR (400 MHz, CDCl₃, Stereoisomer 1): 1.16(9H, s), 1.26 (3H, t, J=7.1), 1.43 (3H, s), 1.88-2.05 (1H, m), 2.14-2.26(1H, m), 2.30-2.50 (2H, m), 4.18 (2H, q, J=7.1), 4.24-4.34 (1H, m), 5.31(1H, brs), 5.51 (1H, d, J=7.7), 6.74 (1H, q, J=7.3). ¹³C NMR (101 MHz,CDCl₃, Stereoisomer 1): 14.23, 19.72, 26.95, 27.57, 36.31, 38.74, 53.33,54.89, 61.85, 89.66, 153.87, 171.61, 176.56, 193.56. ¹H NMR (400 MHz,CDCl₃, Stereoisomer 2): 1.18 (9H, s), 1.26 (3H, t, J=7.1), 1.45 (3H, s),1.88-2.05 (1H, m), 2.14-2.26 (1H, m), 2.30-2.50 (2H, m), 4.18 (2H, q,J=7.1), 4.24-4.34 (1H, m), 5.33 (1H, brs), 5.55 (1H, d, J=7.9), 6.76(1H, q, J=7.3). ¹³C NMR (101 MHz, CDCl₃, Stereoisomer 2): 14.24, 19.74,26.97, 27.80, 36.46, 38.75, 53.48, 54.89, 61.87, 89.81, 153.95, 171.77,176.68, 193.75. Optical rotation: [α]²² _(D)+16.2° (c 0.259, CHCl3). IR(CHCl3): 3428 m, 3358 w, 3116 w, 2982 s, 2874 m, 2110 s, 1740 vs, 1640s, 1510 s, 1480 m, 1393 s (sh), 1377 s, 1349 s, 1284 s, 1232 s, 1025 s,cm⁻¹. ESI MS: 394 ([M+Na]⁺). HR ESI MS: calcd for C₁₆H₂₅O₇N₃Na394.15847; found 394.15886.

Synthesis of ethyl2-(2-amino-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (34)Ethyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (33)

Referring to scheme 7, Fmoc-NH-(L)-Trp-COOH (353 mg, 0.828 mmol, 1.1eq.) and HBTU (343 mg, 0.904 mmol, 1.2 eq.) were suspended in dry DCM (5mL). DIEA (292 mg, 394 μL, 2.26 mmol, 3 eq.) and then the solution of 20(150 mg, 0.753 mmol, 1 eq.) in dry DCM (3 mL) were added by syringe. Thereaction mixture was stirred for 2 h at rt under inert atmosphere. DCM(20 mL) was added and the organic phase was washed with sat. NaHCO₃ (20mL), 1M HCl (20 mL), water (30 mL) and sat. NaCl (30 mL), dried overMgSO₄. DCM was evaporated. The crude product was purified by LC(DCM/EtOAc 2:1, R_(f) 0.21) to give 33 as a light yellow solid (303 mg)in 66% yield. ¹H NMR (400 MHz, CDCl₃): 1.22 (3H, t, J=7.2), 1.82-1.93(1H, m), 2.00-2.26 (3H, m), 3.18 (1H, dd, J=14.5, 7.1), 3.38 (1H, dd,J=14.0, 5.3), 4.09 (2H, q, J=8.2), 4.20 (1H, t, J=7.1), 4.32-4.48 (3H,m), 4.50-4.59 (1H, m), 5.05 (1H, bs), 5.51 (1H, d, J=7.9), 6.61 (1H, d,J=7.5), 7.07 (1H, bs), 7.13 (1H, t, J=7.4), 7.20 (1H, t, J=7.2), 7.30(2H, t, J=7.5), 7.35 (1H, d, J=8.0), 7.40 (2H, t, J=7.5), 7.57 (2H, t,J=6.6), 7.67 (1H, d, J=7.9), 7.76 (2H, d, J=7.6), 8.28 (1H, bs). ¹³C NMR(101 MHz, CDCl₃): 14.20, 27.09, 28.51, 36.20, 47.25, 52.13, 54.86,55.76, 61.77, 67.25, 110.29, 111.39, 118.88, 120.00, 120.10, 120.11,122.41, 123.66, 125.25, 125.29, 126.92, 127.22 (2C), 127.62, 127.86(2C), 136.38, 141.40, 143.87, 143.96, 156.09, 171.28, 171.48, 193.84.Optical rotation: [α]²² _(D)−28.6° (c 0.178, DMF). IR (KBr): 3424 s,3308 m, sh, 2978 w, 2106 m, 1728 s, 1697 m, sh, 1654 m, 1519 m, 1478 w,1450 m, 1382 m, sh, 1376 m, 1343 m, 1289 m, 1224 m, 1104 m-w, 1081 m-w,1040 m, 1032 m, 877 w, 855 w, 760 m, 742 m, 621 m, 427 w, cm⁻¹. ESI MS:630 ([M+Na]⁺). HR ESI MS: calcd for C₃₄H₃₃O₆N₅Na 630.23230; found630.23236.

Ethyl 2-(2-amino-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate(34)

Referring to scheme 7, compound 33 (303 mg, 0.499 mmol, 1 eq.) wasdissolved in dry DCM (5 mL). Piperidine (212 mg, 244 μL, 2.49 mmol, 5eq.) was added by syringe. The reaction mixture was stirred for 4.5 h atrt under inert atmosphere. DCM was evaporated. The crude product waspurified by column LC (CHCl₃/MeOH 20:1 to 10:1, R_(f) 0.29) and acolorless solid 34 (96 mg) was obtained in 50% yield. ¹H NMR (400 MHz,CDCl₃): 1.27 (3H, t, J=7.0), 1.57 (2H, bs), 1.91-2.01 (1H, m), 2.06-2.27(3H, m), 3.05 (1H, dd, J=14.4, 8.1), 3.30 (1H, dd, J=14.4, 4.0), 3.75(1H, dd, J=8.1, 4.2), 4.18 (1H, qd, J=7.1, 2.0), 4.55 (1H, td, J=8.3,4.1), 5.11 (1H, bs), 7.09 (1H, d, J=2.4), 7.12 (1H, ddd, J=8.1, 7.0,1.1), 7.20 (1H, ddd, J=8.1, 7.0, 1.2), 7.37 (1H, dt, J=8.2, 1.0), 7.68(1H, dd, J=7.9, 1.1), 7.88 (1H, d, J=8.3), 8.31 (1H, bs). ¹³C NMR (101MHz, CDCl₃): 14.29, 27.60, 30.82, 36.58, 51.56, 54.77, 55.50, 61.71,111.36, 111.49, 119.23, 119.79, 122.39, 123.45, 127.69, 136.52, 171.92,175.02, 193.81. Optical rotation: [α]²² _(D)−57.6° (c 0.210, DMF). IR(CHCl3): 3479 m, 3355 w, 3215 vw, 3116 w, 2982 m, 2930 m, 2872 w, 2855w, 2110 vs, 1737 s, 1687 s, 1641 m, 1511 m, 1373 m, 1353 m, 1336 m, sh,1191 m, 1115 w, 1092 m, 1009 w, cm⁻¹. ESI MS: 408 ([M+Na]⁺). HR ESI MS:calcd for C₁₉H₂₃O₄N₅Na 408.16423; found 408.16435.

Synthesis of6-diazo-2-((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)-5-oxohexanoicacid triethylammonium salt (35)6-Diazo-2-((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)-5-oxohexanoicacid triethylammonium salt (35)

Referring to scheme 5, compound 20 (238 mg, 1.19 mmol, 1 eq.) wasdissolved in THF (10 mL). 1M solution of NaOH (48 mg, 1.18 mL, 1 eq.)was added and the mixture was stirred for 1 h. Water (4 mL) and 12 (389mg, 1.43 mmol, 1.2 eq.) in THF (10 mL) were added. The solution wasstirred for next 2 h at rt. The reaction was quenched with 1 M formicacid (1.15 mL, 0.95 eq.) and after 5 minutes of stirring the mixture wasevaporated to dryness. The crude product was purified by preparativeHPLC with Et₃N/CH₃COOH buffer. The desired product 35 was obtained as alight orange solid (50 mg) in 13% yield. ¹H NMR (400 MHz, CDCl₃):1.96-2.28 (2H, m), 2.15 (3H, s), 2.37-2.70 (2H, m), 4.40-4.76 (1H, m),4.98 (2H, s).

Synthesis of ethyl6-diazo-2-((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)-5-oxohexanoate (36) Ethyl6-diazo-2-((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)-5-oxohexanoate(36)

Referring to scheme 8, compound 12 (180 mg, 0.663 mmol, 1.1 eq) wassuspended in dry DCM (5 mL). The reaction mixture was cooled to 0° C.and compound 20 (120 mg, 0.602 mmol, 1 eq.) in dry DCM (3 mL) was addedby drop wise. The mixture was stirred for 15 minutes at 0° C. and then 1h at rt. The crude product was purified by column chromatography(EtOAc:hexane, 1:1) and the desired compound 36 was obtained in 65%yield (139 mg) as an yellow oil. ¹H NMR (400 MHz, CDCl₃): 1.27 (3H, t,J=7.1), 1.95-2.06 (1H, m), 2.10-2.27 (1H, m), 2.16 (3H, s), 2.31-2.50(2H, m), 4.19 (2H, q, J=7.1), 4.29 (1H, td, J=8.1, 4.8), 4.82 (2H, dd,J=13.9), 5.29 (1H, brs), 5.72 (1H, d, J=7.7). ¹³C NMR (101 MHz, CDCl₃):9.48, 14.24, 27.18, 36.32, 53.76, 54.58, 55.00, 61.92, 133.90, 140.08,152.30, 155.26, 171.63, 193.52. Optical rotation: [α]²² _(D)+12.2° (c0.229, CHCl3). IR (CHCl3): 3425 m, 3345 w, vbr, 3116 w, 2111 vs, 1838 s,sh, 1820 vs, 1736 vs, 1722 vs, sh, 1640 s, 1511 s, 1229 s, 1200 s, 1114m, 1097 m, 1045 s, cm⁻¹. ESI MS: 378 ([M+Na]⁺). HR ESI MS: calcd forC₁₄H₁₇O₈N₃Na 378.09079; found 378.09102.

Synthesis of isopropyl2-(2-amino-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (38)Isopropyl2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate(37)

Referring to scheme 7, Fmoc-NH-(L)-Trp-COOH (880 mg, 2.06 mmol, 1.1 eq.)and HBTU (854 mg, 2.25 mmol, 1.2 eq.) were suspended in dry DMF (14 mL).DIEA (727 mg, 980 μL, 5.63 mmol, 3 eq.) and then the solution of 3 (400mg, 1.88 mmol, 1 eq.) in dry DMF (5 mL) were added by syringe. Thereaction mixture was stirred for 4 h at rt under inert atmosphere. DMFwas evaporated, DCM (100 mL) was added and the organic phase was washedwith sat. NaHCO₃ (100 mL), water (100 mL), 1M HCl (100 mL), water (100mL) and sat. NaCl (100 mL), dried over MgSO₄. DCM was evaporated. Thecrude product was purified by column chromatography (DCM:EtOAc, 5:1,R_(f) 0.15) to give 37 as a light yellow solid (540 mg) in 46% yield. ¹HNMR (400 MHz, CDCl₃): 1.19 (3H, d, J=6.3), 1.23 (3H, t, J=6.3),1.81-1.92 (1H, m), 2.01-2.28 (3H, m), 3.18 (1H, dd, J=14.5, 7.1), 3.39(1H, dd, J=14.2, 5.2), 4.20 (1H, t, J=7.1), 4.30-4.48 (3H, m), 4.54 (1H,q, J=6.9), 4.88-4.99 (1H, m), 5.07 (1H, bs), 5.50 (1H, d, J=7.9), 6.59(1H, d, J=7.4), 7.07 (1H, bs), 7.14 (1H, t, J=7.0), 7.20 (1H, t, J=7.2),7.30 (2H, tdd, J=7.5, 2.5, 1.1), 7.36 (1H, d, J=8.0), 7.40 (2H, t,J=7.5), 7.56 (2H, t, J=6.6), 7.67 (1H, d, J=7.9), 7.77 (2H, d, J=7.6),8.23 (1H, bs). ¹³C NMR (101 MHz, CDCl₃): 21.78, 21.83, 27.20, 28.47,36.20, 47.23, 52.19, 54.91, 55.75, 67.23, 69.22, 110.28, 111.40, 118.88,120.03, 120.10, 120.12 (2C), 122.45, 123.66, 125.25, 125.30, 127.23(2C), 127.61, 127.86 (2C), 136.35, 141.40 (2C), 143.85, 143.96, 156.09,170.80, 171.42, 193.92. Optical rotation: [α]²² _(D)−32.0° (c 0.193,DMF). IR (KBr): 3424 s, 3300 m, 3130 vw, 2980 w, 2932 s, 2110 m, 1722 s,1695 s, 1654 s, 1625 m, 1547 m, sh, 1532 m, 1520 m, sh, 1478 w, 1385 m,1375 m, 1353 m, 1343 m, 1288 m, 1236 m, 1182 w, 11145 m, 1105 m, 1031m-w, 1010 w, 852 w, 798 w, 758 m, 741 m, 621 w, 427 w-m, cm⁻¹. ESI MS:644 ([M+Na]⁺). HR ESI MS: calcd for C₃₅H₃₅O₆N₅Na 644.24795; found644.24811.

Isopropyl2-(2-amino-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (38)

Referring to scheme 7, compound 37 (500 mg, 0.804 mmol, 1 eq.) wasdissolved in dry DCM (15 mL). Piperidine (342 mg, 393 μL, 4.02 mmol, 5eq.) was added by syringe. The reaction mixture was stirred for 4 h atrt under inert atmosphere. DCM was evaporated. The crude product waspurified by column LC (CHCl₃/MeOH 30:1, R_(f) 0.14) to give 38 as ayellow amorphous solid (170 mg) in 53% yield. ¹H NMR (400 MHz, CDCl₃):1.24 (3H, d, J=6.0), 1.25 (3H, d, J=6.0), 1.50 (2H, bs), 1.88-2.00 (1H,m), 2.04-2.27 (3H, m), 3.04 (1H, dd, J=14.4, 8.2), 3.30 (1H, ddd,J=14.2, 4.2, 0.9), 3.74 (1H, dd, J=8.1, 4.2), 4.51 (1H, td, J=8.3, 4.0),5.02 (1H, hept, J=6.0), 5.11 (1H, bs), 7.08 (1H, d, J=2.3), 7.11 (1H,ddd, J=8.0, 7.1, 1.0), 7.19 (1H, ddd, J=8.1, 7.1, 1.1), 7.36 (1H, d,J=8.1), 7.67 (1H, d, J=7.9), 7.88 (1H, d, J=8.2), 8.45 (1H, bs). ¹³C NMR(101 MHz, CDCl₃): 21.83, 27.65, 30.81, 36.55, 51.64, 54.80, 55.51,69.49, 111.37 (2C), 119.17, 119.72, 122.31, 123.48, 127.67, 136.53,171.42, 175.09, 193.92. Optical rotation: [α]²² _(D)−1.2° (c 0.012,CHCl3). IR (CHCl3): 3311 m, vbr, 2980 w, 2924 w, 2874 w, vs, 2853 w,2104 vs, 1731 s, 1650 s, br, 1618 m, sh, 1512 m, 1388 m, sh, 1375 s,1253 m, 1232 m, 1183 m, 1145 m, 1105 s, 1010 w, 972 vw, 933 vw, 744 m,cm⁻¹. ESI MS: 422 ([M+Na]⁺). HR ESI MS: calcd for C₂₀H₂₅O₄N₅Na422.17988; found 422.17992.

Synthesis of isopropyl6-diazo-2-(((2-methyl-1-(pivaloyloxy)propoxy)carbonyl)amino)-5-oxohexanoate(40) 1-((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)-2-methylpropylpivalate (39)

1-Chloro-2-methylpropyl carbonochloridate (2.00 g, 1.71 mL, 11.69 mmol,1 eq.) was dissolved in dry Et₂O (20 mL). The reaction mixture wascooled to 0° C. and the mix of Et₃N (1.18 g, 1.63 mL, 11.69 mmol, 1 eq.)and EtSH (727 mg, 866 μL, 11.69 mmol, 1 eq.) dissolved in dry Et₂O (10mL) was added by dropwise method during 10 minutes. The reaction mixturewas stirred overnight (23 h) at rt, precipitate was filtered over pad ofcelite and solvent was removed under reduced pressure. The crude productO-(1-chloro-2-methylpropyl)S-ethyl carbonothioate (colorless liquid,2.20 g, 96%) was used for further step without purification.O-(1-Chloro-2-methylpropyl)S-ethyl carbonothioate (1.20 g, 6.10 mmol, 1eq.) was dissolved in pivalic acid (3.74 g, 4.20 mL, 36.61 mmol, 6 eq.)and freshly prepared salt of pivalic acid (1.87 g, 2.10 mL, 18.31 mmol,3 eq.) and DIEA (2.37 g, 3.19 mL, 18.31 mmol, 3 eq.) was added in fewportions. The reaction mixture was heated to 60° C. for 70 h. EtOAc (100mL) was added and the organic phase was extracted with water (50 mL),sat. NaHCO₃ (3×50 mL), sat. NaCl (50 mL), dried over MgSO₄ and solventwas evaporated. The crude product1-(((ethylthio)carbonyl)oxy)-2-methylpropyl pivalate (light yellowliquid, 1.32 g, 83%) was used for further step without purification.1-(((Ethylthio)carbonyl)oxy)-2-methylpropyl pivalate (1.28 g, 4.88 mmol,1 eq.) was dissolved in dry DCM (13 mL), N-hydroxysuccinimide (1.12 g,9.76 mmol, 2 eq.) was added and the suspension was cooled to 0° C.Peracetic acid (1.11 g (100%), 3.09 g (36%), 14.64 mmol, 3 eq., 36%solution in acetic acid) was added by dropwise in 10 minutes. The finalmixture was stirred for 60 minutes at 0° C. and 2 h at rt. DCM (40 mL)was added and the organic phase was washed with water (20 mL) and sat.NaCl (20 mL) and dried over MgSO₄. DCM was evaporated and the productwas purified by LC (hexane/EtOAc 5:3, R_(f) 0.26). The product 39 wasobtained as a light yellow oil (863 mg) in 56% yield (over 3 steps). ¹HNMR (400 MHz, CDCl₃): 1.00 (6H, d, J=6.9), 1.21 (9H, s), 2.08-2.19 (1H,m), 2.81 (4H, s), 6.55 (1H, d, J=5.0). ¹³C NMR (101 MHz, CDCl₃): 16.06,16.38, 25.55, 26.89, 31.84, 39.05, 98.19, 150.37, 168.48, 176.14.Optical rotation: [α]²² _(D)−3.0° (c 0.230, CHCl3). IR (CHCl3): 2978 m,2938 w, 2878 w, 1821 s, 1795 s, 1748 vs, br, 1481 m, 1463 w, 1432 m,1396 w, 1373 m, 1366 m, sh, 1279 m, 1199 s, 1046 m, 998 m, sh, 987 m,932 s cm⁻¹. ESI MS: 338 ([M+Na]⁺). HR ESI MS: calcd for C₁₄H₂₁O₇NNa338.12102; found 338.12115.

Isopropyl6-diazo-2-(((2-methyl-1-(pivaloyloxy)propoxy)carbonyl)amino)-5-oxohexanoate(40)

Compound 39 (399 mg, 1.27 mmol, 0.9 eq) was suspended in dry DCM (7 mL).The reaction mixture was cooled to 0° C. and compound 3 (300 mg, 1.41mmol, 1 eq.) in dry DCM (3 mL) was added by drop wise. The mixture wasstirred for 15 minutes at 0° C. and then 2 h at rt. The crude productwas purified by column chromatography (EtOAc/hexane 1:2, R_(f) 0.29 and0.31) and the desired compound 40 was obtained in 54% yield (285 mg) asa yellow oil (mixture of two stereoisomers 1:1). ¹H NMR (400 MHz, CDCl₃,stereoisomer 1): 0.94 (6H, d, J=6.8), 1.16 (9H, s), 1.23 (6H, t, J=6.3),1.83-2.50 (4H, m), 4.22-4.31 (1H, m), 5.02 (1H, hept, J=6.8), 5.29 (1H,bs), 5.48 (1H, d, J=8.3), 6.52 (1H, d, J=4.9). ¹³C NMR (101 MHz, CDCl₃,stereoisomer 1): 16.40, 16.54, 27.00, 28.05, 31.87, 36.29, 38.96, 53.38,54.82, 69.64, 94.21, 154.28, 171.31, 176.56, 193.87. ¹H NMR (400 MHz,CDCl₃, stereoisomer 2): 0.93 (6H, d, J=6.8), 1.18 (9H, s), 1.22 (6H, t,J=6.3), 1.83-2.50 (4H, m), 4.22-4.31 (1H, m), 5.00 (1H, sept, J=6.8),5.37 (1H, bs), 5.45 (1H, d, J=8.3), 6.48 (1H, d, J=4.9). ¹³C NMR (101MHz, CDCl₃, stereoisomer 2): 16.37, 16.54, 26.98, 27.74, 31.91, 36.55,38.92, 53.54, 54.82, 69.66, 93.87, 154.22, 171.17, 176.81, 193.58.Optical rotation: [α]²² _(D)+11.5° (c 0.261, CHCl3). IR (CHCl3): 3428 m,3116 w, 2982 s, 2936 m, 2878 m, 2110 vs, 1741 vs, br, 1731 vs, sh, 1641s, 1508 s, 1480 m, 1463 m, 1400 m, sh, 1385 s, sh, 1377 s, 1365 s, sh,1281 s, 1231 s, 1183 m, 1146 s, 1105 s, 990 s, 941 m cm⁻¹. ESI MS: 436([M+Na]⁺). HR ESI MS: calcd for C₁₉H₃₁O₇N₃Na 436.20542; found 436.20553.

Synthesis of isopropyl6-diazo-5-oxo-2-((((pivaloyloxy)methoxy)carbonyl)amino) hexanoate (42)((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl pivalate (41)

Chloromethyl carbonochloridate (2.00 g, 1.38 mL, 15.51 mmol, 1 eq.) wasdissolved in dry Et₂O (20 mL). The reaction mixture was cooled to 0° C.and the mix of Et₃N (1.57 g, 2.16 mL, 15.51 mmol, 1 eq.) and EtSH (964mg, 1.15 mL, 15.51 mmol, 1 eq.) dissolved in dry Et₂O (5 mL) was addedby dropwise method during 5 minutes. The reaction mixture was stirredovernight (25 h) at rt, precipitate was filtered over pad of celite andsolvent was removed under reduced pressure. The crude product0-(chloromethyl)S-ethyl carbonothioate (colorless liquid) was used forfurther step without purification. 0-(Chloromethyl)S-ethylcarbonothioate (2.40 g, 15.51 mmol, 1 eq.) was dissolved in pivalic acid(9.51 g, 93.14 mmol, 6 eq.) and freshly prepared salt of pivalic acid(4.76 g, 46.57 mmol, 3 eq.) and DIEA (6.02 g, 8.1 mL, 46.57 mmol, 3 eq.)was added in few portions. The reaction mixture was heated to 60° C. for22 h. EtOAc (100 mL) was added and the organic phase was extracted withwater (100 mL), sat. NaHCO₃ (3×100 mL), sat. NaCl (100 mL), dried overMgSO₄ and solvent was evaporated. The crude product(((ethylthio)carbonyl)oxy)methyl pivalate (light yellow liquid, 3.30 g,97%) was used for further step without purification.(((Ethylthio)carbonyl)oxy)methyl pivalate (3.20 g, 14.53 mmol, 1 eq.)was dissolved in dry DCM (40 mL), N-hydroxysuccinimide (3.34 g, 29.05mmol, 2 eq.) was added and the suspension was cooled to 0° C. Peraceticacid (3.31 g (100%), 9.21 g (36%), 43.58 mmol, 3 eq., 36% solution inacetic acid) was added by dropwise in 15 minutes. The final mixture wasstirred for 60 minutes at 0° C. and 2 h at rt. DCM (50 mL) was added andthe organic phase was washed with water (30 mL) and sat. NaCl (30 mL)and dried over MgSO₄. DCM was evaporated and the product was purified byLC (hexane:EtOAc, 2:1, R_(f) 0.27). The product 41 was obtained as acolorless solid (2.54 g) in 64% yield (over 3 steps). ¹H NMR (400 MHz,CDCl₃): 1.24 (9H, s), 2.84 (4H, s), 5.86 (2H, s). ¹³C NMR (101 MHz,CDCl₃): 25.56 (2C), 26.86 (3C), 38.96, 83.67, 150.90, 168.34 (2C),176.54. IR (CHCl3): 2979 m, 2939 w, 2876 w, 1823 s, 1796 vs, 1649 vs,1481 m, 1463 m, 1456 w, 1431 m, 1398 w, 1371 m, 1367 m, 1280 m, 1199 vs,1110 vs, 1047 m, 998 s, 986 s, 942 m, sh, 924 s, 853 w, cm⁻¹. ESI MS:296 ([M+Na]⁺). HR ESI MS: calcd for C₁₁H₁₅O₇NNa 296.07407; found296.07410.

Isopropyl 6-diazo-5-oxo-2-((((pivaloyloxy)methoxy)carbonyl)amino)hexanoate (42)

Referring to scheme 6, compound 41 (320 mg, 1.17 mmol, 1.0 eq) wassuspended in dry DCM (6 mL). The reaction mixture was cooled to 0° C.and NH₂-DON—COOEt (250 mg, 1.17 mmol, 1 eq.) in dry DCM (3 mL) was addedby drop wise. The mixture was stirred for 15 minutes at 0° C. and then 2h at rt. The crude product was purified by column chromatography(EtOAc:hexane, 1:2, R_(f) 0.21) and the desired compound 42 was obtainedin 40% yield (175 mg) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): 1.19(9H, s), 1.23 (3H, d, J=6.2), 1.24 (3H, d, J=6.2), 1.90-2.05 (1H, m),2.14-2.25 (1H, m), 2.31-2.51 (2H, m), 4.28 (1H, td, J=8.2, 4.7), 5.03(1H, hept, J=6.2), 5.27 (1H, bs), 5.65 (1H, d, J=8.1), 5.69 (1H, d,J=5.7), 5.73 (1H, d, J=5.7). ¹³C NMR (101 MHz, CDCl₃): 21.79, 21.81,26.97, 36.34, 38.86, 53.63, 54.90, 69.75, 80.33, 154.40, 171.01, 177.51,193.43. Optical rotation: [α]²² _(D)+13.0° (c 0.184, CHCl3). IR (CHCl3):3424 m, 3354 w, br, 3116 w, 2984 s, 2937 m, 2875 s, 2110 vs, 1747 vs,1730 vs, sh, 1642 s, 1512 s, 1481 m, 1466 m, 1453 m, 1377 s, 1282 s,1182 m, 1145 s, 1105 s, 994 s, 942 m, cm⁻¹. ESI MS: 394 ([M+Na]⁺). HRESI MS: calcd for C₁₆H₂₅O₇N₃Na 394.15847; found 394.15855.

Synthesis of isopropyl6-diazo-5-oxo-2-((((2-(pivaloyloxy)propan-2-yl)oxy)carbonyl) amino)hexanoate (44) 2-(((4-Nitrophenyloxy)carbonyl)oxy)propan-2-yl pivalate(43)

2-Chloropropan-2-yl (4-nitrophenyl) carbonate (prepared by a methodreported in US 2006/229361 A1, 300 mg, 1.16 mmol, 1 eq.) was dissolvedin dry DCM (15 mL). Mercury pivalate (559 mg, 1.39 mmol, 1.2 eq.) wasadded an d reaction mixture was stirred overnight (19 h) at rt underinert. The solid precipitate (HgCl₂) was removed by filtration, DCM (15mL) was added and RM was extracted with sat. NaHCO₃ (15 mL), sat brine(15 mL), dried over MgSO₄ and solvent was evaporated. Product 43 wasobtained as a light yellow oil (301 mg) in 80% yield. ¹H NMR (400 MHz,CDCl₃): 1.20 (9H, s), 1.91 (6H, s), 7.34-7.40 (2H, m), 8.24-8.30 (2H,m). ¹³C NMR (101 MHz, CDCl₃): 25.28 (2C), 27.02 (3C), 39.59, 107.71,121.98 (2C), 125.40 (2C), 145.50, 149.07, 155.41, 175.97. IR (CHCl3):3031 w, 2976 w, 2875 w, 1777 m, 1736 m, 1618 w, 1595 w, 1528 m-s, 1493m, 1481 w, 1439 w, 1396 w, 1376 w, 1349 m, 1322 w, 1264 m, 1191 m, 1112vs, 1094 s, sh, 1030 w, 980 w, 859 m, 682 vw, 491 vw cm⁻¹. ESI MS: 348([M+Na]⁺). HR ESI MS: calcd for C₁₅H₁₉O₇NNa 348.10537; found 348.10543.

Isopropyl 6-diazo-5-oxo-2-((((2-(pivaloyloxy)propan-2-yl)oxy)carbonyl)amino) hexanoate (44)

Compound 43 (48 mg, 0.148 mmol, 1 eq.) was dissolved in dry DMF (2 mL)and reaction mixture was cooled to 0° C. Compound 3 (79 mg, 0.369 mmol,2.5 eq.) dissolved in dry DMF (1 mL) was added by syringe. Reactionmixture was stirred at 0° C. under inert overnight for 3 h. DMF wasevaporated and the crude mixture was purified by LC (hexane:EtOAc, 2:1).Product 44 was obtained as a light yellow oil (49 mg) in 83% yield. ¹HNMR (400 MHz, CDCl₃): 1.19 (9H, s), 1.23 (3H, d, J=6.3), 1.24 (3H, d,J=6.3), 1.80 (3H, s), 1.83 (3H, s), 1.90-2.01 (1H, m), 2.14-2.27 (1H,m), 2.29-2.51 (2H, m), 4.24 (1H, dt, J=8.3, 4.7), 5.05 (1H, hept,J=6.3), 5.31 (1H, bs), 5.44 (1H, d, J=8.2). ¹³C NMR (101 MHz, CDCl₃):21.80, 21.82, 25.76, 25.91, 27.07 (3C), 27.79, 36.46, 39.48, 53.26,54.87, 69.59, 105.44, 153.16, 171.31, 176.21, 193.63. Optical rotation:[α]²² _(D)+12.8° (c 0.133, CHCl3). IR (CHCl3): 3430 w, 3116 w, 2984 m,2936 m, 2874 m, 2110 s, 1732 vs, br, 1641 m, 1502 s, 1481 m, 1466 m,1462 m, 1455 m, 1452 m, 1397 m, sh, 1384 s, 1374 s, 1365 s, sh, 1198 s,1184 s, 1147 m, sh, 1128 s, 1112 s, 1105 s, 1045 m, 942 w cm⁻¹.

Synthesis of isopropyl6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino) hexanoate(47) Chloro(phenyl)methyl (4-nitrophenyl) carbonate (45)

Chloro(phenyl)methyl carbonochloridate (prepared by US20110319422, 900mg, 4.39 mmol, 1 eq.) was dissolved in dry DCM (20 mL). 4-Nitrophenol(611 mg, 4.39 mmol, 1 eq.) was added and the mixture was cooled to 0° C.Pyridine (347 mg, 355 μL, 4.39 mmol, 1 eq.) dissolved in dry DCM (5 mL)was added by dropwise method during 5 minutes. Reaction mixture wasstirred for 2 h at rt. DCM was evaporated and the crude product waspurified by LC (DCM:hexane, 1:1). The product 45 was obtained as acolorless solid (520 mg) in 39% yield. ¹H NMR (400 MHz, CDCl₃): 7.33(1H, s), 7.41-7.50 (5H, m), 7.58-7.63 (2H, m), 8.28-8.34 (2H, m). ¹³CNMR (101 MHz, CDCl₃): 87.37, 121.83 (2C), 125.59 (2C), 126.41 (2C),129.07 (2C), 130.56, 136.35, 145.91, 150.50, 155.07. Optical rotation:[α]²² _(D)−0.9° (c 0.318, CHCl3). IR (CHCl3): 3119 w, 3088 w, 3071 vw,3032 w, 1788 vs, 1772 s, sh, 1619 m, 1595 m, 1530 vs, 1492 s, 1456 m,1349 vs, 1317 m, 1296 m, 1232 vs, sh, 1178 m, sh, 1165 m, 1111 m, 1105w, sh, 1078 m, 1054 s, 1029 m, 1014 m, 1002 w, 978 s, 920 w, 872 s, 854s, 830 vw, 708 s, 695 m, sh, 680 w, 626 vw, 618 vw, 530 vw, 495 w, 403 wcm⁻¹. ESI MS: 329 ([M+Na]⁺). HR ESI MS: calcd for C₁₄H₁₀O₅NClNa330.01397; found 330.01367.

(((4-Nitrophenyloxy)carbonyl)oxy)(phenyl)methyl pivalate (46)

Compound 45 (100 mg, 0.325 mmol, 1 eq.) and mercury pivalate (157 mg,0.390 mmol, 1.2 eq.) were dissolved in dry DCM (6 mL). Reaction mixturewas stirred at rt under inert overnight (16 h). DCM (10 mL) was addedand reaction mixture was washed with sat. NaHCO₃ (10 mL) and brine (10mL), organic phase was dried over MgSO₄ and DCM was evaporated. Theproduct 46 (115 mg) was obtained in 95% yield and was used for furtherstep without any purification. ¹H NMR (400 MHz, CDCl₃): 1.28 (9H, s),7.38-7.43 (2H, m), 7.44-7.50 (3H, m), 7.57-7.60 (1H, m), 7.61 (1H, s),8.23-8.33 (2H, m). ¹³C NMR (101 MHz, CDCl₃): 27.02 (3C), 39.11, 93.80,121.86 (2C), 125.47 (2C), 126.84 (2C), 128.97 (2C), 130.48, 134.39,145.69, 150.73, 155.32, 176.44. Optical rotation: [α]²² _(D)−6.0° (c0.201, CHCl3). IR (CHCl3): 3118 w, 3087 w, 3072 w, 3031 m, 2980 m, 2875w, 1775 vs, 1747 s, 1618 m, 1595 m, 1529 vs, 1493 s, 1480 m, 1459 m,1399 m, 1365 m, 1349 vs, 1279 vs, 1248 vs, 1165 s, 1123 vs, 1112 s, sh,1030 s, 1014 m, 1003 m, 970 s, br, 943 s, 918 m, 865 s, 860 s, 832 w,697 s, 682 w, 633 w, 619 vw, 530 vw, 495 w, 403 vw cm⁻¹. ESI MS: 396([M+Na]⁺). HR ESI MS: calcd for C₁₉H₁₉O₇NNa 396.10537; found 396.10546.

Isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (47)

Compound 46 (115 mg, 0.308 mmol, 1 eq.) was dissolved in dry DCM (3 mL).DON iPr ester (72 mg, 0.339 mmol, 1.1 eq.) dissolved in dry DCM (2 mL)was added by syringe. Reaction mixture was stirred at rt under inertovernight (24 h) under inert. Further DONiPr ester (72 mg, 0.339 mmol,1.1 eq.) dissolved in dry DCM (2 mL) was added and stirring wascontinued for next 24 h. DCM was evaporated and the crude mixture waspurified by preparative HPLC (AcN/H₂O, HCOOH). Product 47 was obtainedas a light brown oil (66 mg) in 48% yield. ¹H NMR (400 MHz, CDCl₃):1.17-1.31 (15H, m), 1.90-2.06 (1H, m), 2.12-2.31 (1H, m), 2.31-2.54 (2H,m), 4.27-4.36 (1H, m), 5.03 (1H, hept, J=6.3), 5.29 (1H, bs), 5.59 (1H,d, J=8.1), 7.36-7.42 (3H, m), 7.46-7.51 (2H, m), 7.61 (s, 1H). ¹³C NMR(101 MHz, CDCl₃): 21.82 (2C), 26.99 (3C), 27.70, 36.35, 38.99, 53.55,54.90, 69.79, 90.93, 126.57 (2C), 128.66 (2C), 129.64, 135.95, 153.82,171.10, 176.33, 193.51. Optical rotation: [α]²² _(D)+12.5° (c 0.246,CHCl3). IR (CHCl3): 3425 w, 3116 w, 3098 vw, 3070 vw, 3029 m, 2984 m,2937 m, 2875 w, 2110 s, 1735 vs, br, 1641 s, 1590 w, 1507 s, 1480 m,1457 m, 1398 m, 1377 s, 1367 s, sh, 1366 s, sh, 1280 s, 1182 m, 1146 s,sh, 1133 s, 1105 s, 1085 m, 1057 s, 1027 s, 1003 m, 942 m, 918 w, 697 m,619 vw cm⁻¹. ESI MS: 470 ([M+Na]⁺).

Synthesis of isopropyl6-diazo-2-((((isobutyryloxy)methoxy)carbonyl)amino)-5-oxohexanoate (49)((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl isobutyrate (48)

Chloromethyl carbonochloridate (1.00 g, 690 μL, 7.76 mmol, 1 eq.) wasdissolved in dry Et₂O (10 mL). The reaction mixture was cooled to 0° C.and a mixture of Et₃N (785 mg, 1081 μL, 7.76 mmol, 1 eq.) and EtSH (482mg, 574 μL, 7.76 mmol, 1 eq.) dissolved in dry Et₂O (3 mL) was added bydropwise method over 5 minutes. The reaction mixture was stirredovernight (18 h) at rt, precipitate was filtered over pad of celite andsolvent was removed under reduced pressure. The crude product-(chloromethyl)S-ethyl carbonothioate (colorless liquid) was used forfurther step without purification. 0-(Chloromethyl)S-ethylcarbonothioate (1.10 g, 7.11 mmol, 1 eq.) was dissolved in isobutyricacid (1.88 g, 1. 94 mL, 21.35 mmol, 3 eq.) and freshly prepared salt ofisobutyric acid (1.88 g, 1.94 mL, 21.35 mmol, 3 eq.) and DIEA (2.76 g,3.81 mL, 21.35 mmol, 3 eq.) was added in few portions. The reactionmixture was heated to 60° C. for 20 h. EtOAc (50 mL) was added and theorganic phase was extracted with water (50 mL), sat. NaHCO₃ (3×50 mL),sat. NaCl (50 mL), dried over MgSO₄ and solvent was evaporated. Thecrude product (((ethylthio)carbonyl)oxy)methyl isobutyrate (light yellowliquid, 1.14 g, 72%) was used for further step without purification.(((Ethylthio)carbonyl)oxy)methyl isobutyrate (1.12 g, 5.04 mmol, 1 eq.)was dissolved in dry DCM (15 mL), N-hydroxysuccinimide (1.16 g, 10.08mmol, 2 eq.) was added and the suspension was cooled to 0° C. Peraceticacid (1.15 g (100%), 3.19 g (36%), 15.12 mmol, 3 eq., 36% solution inacetic acid) was added by dropwise in 10 minutes. The final mixture wasstirred for 60 minutes at 0° C. and 2 h at rt. DCM (20 mL) was added andthe organic phase was washed with water (15 mL) and sat. NaCl (15 mL)and dried over MgSO₄. DCM was evaporated and the product was purified byLC (hexane:EtOAc, 2:1, R_(f) 0.24). The product 48 was obtained as acolorless oil (842 mg) in 64% yield (over 3 steps). ¹H NMR (400 MHz,CDCl₃): 1.19 (6H, d, J=7.0), 2.63 (1H, hept, J=7.0), 2.83 (4H, s), 5.85(2H, s). ¹³C NMR (101 MHz, CDCl₃): 18.60, 25.54, 33.77, 83.50, 150.92,168.39, 175.08. IR (CHCl3): 2981 m, 2945 w, 2880 w, 1823 s, 1795 vs,1748 vs, br, 1720 m, sh, 1471 m, 1431 m, 1389 w, 1370 m, 1231 vs, 1199vs, 1113 s, 1045 m, 925 s, cm⁻¹. ESI MS: 282 ([M+Na]⁺). HR ESI MS: calcdfor C₁₀H₁₃O₇NNa 282.05842; found 282.05848.

Isopropyl6-diazo-2-((((isobutyryloxy)methoxy)carbonyl)amino)-5-oxohexanoate (49)

Compound 48 (268 mg, 1.03 mmol, 1.1 equiv) was dissolved in absolutedichloromethane (8 mL). This solution was cooled to 0° C. and a solutionof the compound 3 (200 mg, 0.94 mmol), in dichloromethane (1 mL) wasadded dropwise. The reaction mixture was stirred for 15 min at 0° C. ina cooling bath. The reaction mixture was then stirred at roomtemperature for 1 h. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform:acetone, 10:1) toeffort the desired product 49 (180 mg, 54%) as a yellow amorphous solid.¹H NMR (400 MHz, CDCl₃): 1.17-1.19 (6H, m), 1.25-1.27 (6H, m), 1.94-2.04(1H, m), 2.18-2.26 (1H, m), 2.32-2.49 (2H, m), 2.59 (1H, hept, J=6.9),4.31 (1H, td, J=8.1, 4.7), 5.05 (1H, hept, J=6.3), 5.28 (1H, s), 5.62(1H, d, J=8.0), 5.71 (1H, d, J=5.8), 5.75 (1H, d, J=5.8). ¹³C NMR (101MHz, CDCl₃): 18.82 (2C), 21.84, 21.86, 27.67, 33.91, 36.37, 53.66,54.99, 69.84, 80.14, 154.45, 171.03, 176.19. IR (CHCl3):3424 w, 2111 s,vs, 1750 sh, vs, 1732 vs, 1641 m, s, 1512 s, 1387 sh, s, 1377 s, 1370sh, s cm⁻¹. Optical rotation: [α]²² _(D)+5.4° (c 0.202, CH₂C12). ESI MS:380 ([M+Na]⁺). HR ESI MS: calcd for C₁₅H₂₃O₇N₃Na 380.14282; found380.14286.

Synthesis of isopropyl2-((((4-(2-(2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate (51) Isopropyl2-((((4-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate(50)

9-Fluorenylmethyloxycarbonyl-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl)carbonate (191 mg, 0.249 mmol, 1.2 equiv.), was dissolved in dry DMF(2.0 mL) and a solution of compound 3 (44 mg, 0.208 mmol) in dry DMF(1.0 mL) was added dropwise. To this reaction mixture was addeddiisopropylethyl amine (130 μL, 0.747 mmol, 3 equiv.) dropwise. Thereaction mixture was stirred at room temperature overnight. The organicsolvent was evaporated in vacuo. The residue was chromatographed onsilica gel (chloroform:methanol, 15:1) to yield the desired product 50(110 mg, 53%) as a white amorphous solid. ¹H NMR (400 MHz, DMSO): 0.85(3H, d, J=6.8), 0.88 (3H, d, J=6.9), 1.16 (3H, d, J=6.6), 1.18 (3H, d,J=6.6), 1.31-1.49 (2H, m), 1.54-1.81 (3H, m), 1.89-2.03 (2H, m),2.35-2.44 (2H, m), 2.89-3.05 (2H, m), 3.91-3.98 (2H, m), 4.20-4.34 (3H,m), 4.39-4.44 (1H, m), 4.86-5.00 (3H, m), 5.40 (2H, s), 5.97 (1H, t,J=5.9), 6.05 (1H, s), 7.28 (2H, d, J=8.7), 7.32 (2H, td, J=7.5, 1.2),7.39-7.44 (3H, m), 7.59 (2H, d, J=8.3), 7.66 (1H, d, J=7.7), 7.74 (2H,t, J=7.8), 7.89 (2H, d, J=7.6), 8.12 (1H, d, J=7.5), 10.06 (1H, s). ¹³CNMR (101 MHz, DMSO): 18.28, 19.23, 21.45, 21.52, 25.78, 26.80, 29.48,30.46, 36.32, 38.58, 46.69, 53.10, 53.42, 60.08, 65.29, 65.69, 68.04,118.91 (2C), 120.10 (2C), 125.37 (2C), 127.07 (2C), 127.65 (2C), 128.61(2C), 131.65, 138.64, 140.71 (2C), 143.77, 143.90, 156.13 (2C), 158.90,170.60, 171.28, 171.55, 194.03. IR (KBr): 3400 s, br, sh, 3327 s, br,3066 w, 2964 m, 2937 m, 2106 s, 1705 vs, br, 1651 vs, 1609 s, 1533 vs,1517 vs, sh, 1479 m, 1466 m, 1450 s, 1415 m, 1386 s, sh, 1376 s, 1334 s,1320 s, sh, 1248 s, 1183 m, 1145 m, 1106 s, 1047 m, 1020 m, sh, 826 w,777 w, sh, 621 w, 427 w cm⁻¹. Optical rotation: [α]²² _(D)−15.6° (c0.631, DMSO). ESI MS: 863 ([M+Na]⁺). HR ESI MS: calcd for C₄₃H₅₂H₈O₁₀Na863.36986; found 863.36997.

Isopropyl 2-((((4-(2-(2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate (51)

Compound 50 (110 mg, 0.131 mmol) was dissolved in dry DMF (0.5 mL).Piperidine (32 μL 0.327 mmol, 2.5 equiv.) was added and the reactionmixture was stirred at room temperature for 4 h. The organic solvent wasevaporated in vacuo. The residue was chromatographed on silica gel(chloroform:methanol, 2:1) to afford the desired product 51 (70 mg, 87%)as a white amorphous solid.

Synthesis of isopropyl2-((((4-(2-(2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate (52) Isopropyl2-((((4-(2-(2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-6-diazo-5-oxohexanoate (52)

Compound 51 (100 mg, 0.162 mmol), was dissolved in dry DMF (2 mL) and tothis solution was added diisopropylethyl amine (144 μL, 0.842 mmol, 5.1equiv.) was added dropwise followed by acetanhydride (76.5 μL, 0.81mmol, 5.0 equiv.). The reaction mixture was stirred at room temperaturefor 2 h. The organic solvent was evaporated in vacuo. The residue waschromatographed on silica gel (chloroform:methanol, 7:1) to afford thedesired product 52 (97 mg, 91%) as a yellow amorphous solid.

Synthesis of Isopropyl2-(2-(2-amino-3-methylbutanamido)-5-ureidopentanamido)-6-diazo-5-oxohexanoate(56) Isopropyl2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-6-diazo-5-oxohexanoate(53)

Fmoc-Cit-COOH (2.05 g, 5.16 mmol, 1.1 equiv.) and TBTU (1.81 g, 5.63mmol, 1.2 equiv.) were solved in absolute DMF (40 mL) anddiisopropylethyl amine (2.51 mL, 14.07 mmol, 3 equiv.) was added. Thereaction mixture was stirred at room temperature for 30 min. and thenthe solution of 3 (1.0 g, 4.69 mmol) in absolute DMF (20 mL) were addedby syringe. The reaction mixture was stirred for 2 h at room temperatureunder inert atmosphere. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform:methanol, 15:1) toafford the desired product 53 (1.84 g, 66%) as a yellow amorphous solid.¹H NMR (400 MHz, DMSO): 1.16 (3H, d, J=4.6), 1.17 (3H, d, J=4.4),1.37-1.57 (3H, m), 1.61-1.70 (1H, m), 1.76-1.86 (1H, m), 1.94-2.03 (1H,m), 2.33-2.46 (2H, m), 2.94-3.03 (2H, m), 4.03-4.08 (1H, m), 4.15-4.31(4H, m), 4.88 (1H, hept, J=6.3), 5.42 (2H, s), 5.96 (1H, t, J=5.8), 6.01(1H, s), 7.31-7.34 (2H, m), 7.39-7.43 (2H, m), 7.53 (1H, d, J=8.1),7.73-7.74 (2H, m), 7.88 (2H, d, J=7.5), 8.28 (1H, d, J=7.5). ¹³C NMR(101 MHz, DMSO): 21.45, 21.49, 25.91, 26.72, 29.39, 36.22, 38.77, 46.68,51.48, 54.06, 65.67, 68.02, 120.10 (2C), 125.36 (2C), 127.09 (2C),127.65 (2C), 140.72 (2C), 143.80, 143.91, 155.96, 158.87, 171.05,172.29, 194.04. IR (KBr): 3435 vs, br, 3348 vs, br, sh, 3068 m, 2979 m,2936 m, 2871 w, 2105 s, 1723 s, 1678 vs, br, 1610 s, sh, 1540 s, br,1478 s, 1466 m, sh, 1450 m, 1386 s, sh, 1376 s, 1252 s, 1220 m, sh, 1184m, sh, 1146 m, 1105 s, 1052 m, 1032 m, 760 m, 741 m, 621 m, 427 s cm⁻¹.ESI MS: 615 ([M+Na]⁺). HR ESI MS: calcd for C₃₀H₃₆O₇N₆Na 615.25377;found 615.25383.

Isopropyl 2-(2-amino-5-ureidopentanamido)-6-diazo-5-oxohexanoate (54)

Compound 53 (1.84 g, 3.11 mmol), was dissolved in dry DMF (24 mL).Piperidine (767 μL 7.76 mmol, 2.5 equiv.) was added and the reactionmixture was stirred at room temperature for 4 h. The organic solvent wasevaporated in vacuo. The residue was chromatographed on silica gel(chloroform:methanol, 2:1) to afford the desired product 54 (874 mg,76%) as a white amorphous solid. ¹H NMR (400 MHz, DMSO): 1.17 (3H, d,J=4.4), 1.18 (3H, d, J=4.3), 1.27-1.47 (3H, m), 1.48-1.58 (1H, m),1.76-1.85 (1H, m), 1.93-2.01 (1H, m), 2.31-2.43 (2H, m), 2.93 (2H, q,J=6.4), 3.14 (1H, dd, J=7.3, 5.3), 4.88 (1H, hept, J=6.3), 5.36 (2H, s),5.90 (1H, t, J=5.7), 6.07 (1H, s), 8.13 (1H, d, J=7.7). ¹³C NMR (101MHz, DMSO): 21.45, 21.50, 26.08, 26.48, 32.69, 36.25, 51.22, 54.21,68.03, 158.72, 171.18, 175.56, 194.05. IR (CHCl3): 3509 w, 3414 w, sh,3446 w, 3357 m, br, sh, 3116 w, 2939 w, 2110 s, 1731 s, 1664 vs, br,1598 m, 1467 w, 1450 m, 1387 m, sh, 1377 s, 1349 m, 1183 w, 1145 m cm⁻¹.Optical rotation: [α]²² _(D)-3.2° (c 0.218, CH₂C12). ESI MS: 371([M+H]⁺). HR ESI MS: calcd for C₃H₄₀O₁₅N₃Na 393.18569; found 393.18575.

Isopropyl11-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-8-(3-ureidopropyl)-2-oxa-4,7,10-triazadodecan-12-oate(55)

Fmoc-Val-COOH (484 mg, 1.43 mmol, 1.1 equiv.) and TBTU (499 mg, 1.55mmol, 1.2 equiv.) were solved in absolute DMF (10 mL) anddiisopropylethyl amine (677 μL, 3.89 mmol, 3 equiv.) was added. Thereaction mixture was stirred at room temperature for 30 min. and thenthe solution of compound 54 (480 mg, 1.30 mmol) in absolute DMF (5 mL)were added by syringe. The reaction mixture was stirred for 3 h at roomtemperature under inert atmosphere. The organic solvent was evaporatedin vacuo. The residue was chromatographed on silica gel(chloroform:methanol, 15:1) to afford the desired product 55 (750 mg,84%) as a white amorphous solid. ¹H NMR (400 MHz, DMSO): 0.84 (3H, d,J=6.7), 0.86 (3H, d, J=6.8), 1.16 (3H, d, J=4.5), 1.17 (3H, d, J=4.5),1.32-1.54 (3H, m), 1.60-1.68 (1H, m), 1.73-1.82 (1H, m), 1.91-2.04 (2H,m), 2.30-2.46 (2H, m), 2.90-3.02 (2H, m), 3.90 (1H, dd, J=9.1, 7.0),4.13-4.18 (1H, m), 4.20-4.34 (4H, m), 4.87 (1H, hept, J=6.2), 5.41 (2H,s), 5.94 (1H, t, J=5.8), 6.04 (1H, s), 7.30-7.34 (2H, m), 7.39-7.45 (3H,m), 7.72-7.76 (2H, m), 7.89 (2H, d, J=7.5), 8.01 (1H, d, J=7.6), 8.31(1H, d, J=7.4). ¹³C NMR (101 MHz, DMSO): 18.26, 19.26, 21.48, 21.52,25.99, 26.57, 29.54, 30.51, 36.09, 38.79, 46.71, 51.37, 52.15, 60.05,65.68, 68.04, 120.15 (2C), 125.41 (2C), 127.12 (2C), 127.68 (2C),140.72, 140.75, 143.79, 143.93, 156.12, 158.84, 171.03, 171.11, 171.76,194.15. IR (KBr): 3415 m, vbr, sh, 3360 m, br, 3283 m, 3068 w, 2964 w,2937 w, 2873 w, 2106 m, 1727 m, 1686 s, 1655 vs, sh, 1645 vs, 1540 s,br, 1478 w, 1465 m, 1451 m, 1386 m, sh, 1376 m, 1293 m, 1249 m, 1226 m,1183 w, sh, 1146 m, 1106 m, 1033 w, 1009 vw, 760 w, 741 w, 621 vw, 427vw cm⁻¹. Optical rotation: [α]²² _(D)−19.3° (c 0.114, DMSO). ESI MS: 714([M+Na]⁺). HR ESI MS: calcd for C₃₅H₄₅N₇O₈Na 714.32218; found 714.32218.

Isopropyl2-(2-(2-amino-3-methylbutanamido)-5-ureidopentanamido)-6-diazo-5-oxohexanoate(56)

Compound 55 (200 mg, 0.289 mmol), was dissolved in dry DMF (3 mL).Piperidine (71 μL 0.723 mmol, 2.5 equiv.) was added and the reactionmixture was stirred at room temperature for 4 h. The organic solvent wasevaporated in vacuo. The residue was chromatographed on silica gel(chloroform:methanol, 2:1) to afford the desired product 56 (110 mg,81%) as a white amorphous solid. ¹H NMR (400 MHz, DMSO): 0.77 (3H, d,J=6.8), 0.87 (3H, d, J=6.9), 1.16 (3H, d, J=5.3), 1.18 (3H, d, J=5.2),1.30-1.52 (3H, m), 1.57-1.68 (1H, m), 1.73-1.83 (1H, m), 1.86-2.01 (2H,m), 2.34-2.44 (2H, m), 2.89-3.00 (2H, m), 4.06-4.18 (2H, m), 4.29-4.37(1H, m), 4.88 (1H, hept, J=6.3), 5.37 (2H, s), 5.91 (1H, t, J=5.8), 6.05(1H, s), 8.04 (1H, d, J=7.8), 8.34 (1H, d, J=7.4). ¹³C NMR (101 MHz,DMSO): 17.49, 18.76, 21.46, 21.50, 25.86, 26.44, 29.61, 30.44, 36.08,38.64, 51.45, 51.93, 57.96, 68.04, 158.99, 169.93, 170.95, 171.58,194.03. IR (KBr): 3500 w, br, sh, 3338 m, vbr, 3116 w, 2984 s 2965 s,2936 m, 2874 m, 2109 s, 1731 s, 1653 vs, br, 1602 s, br, sh, 1552 s, br,1517 s, 1466 m, 1452 m, 1387 s, sh, 1376 s, 1234 s, 1183 m, 1145 s, 1106s cm⁻¹. Optical rotation: [α]²² _(D)−13.0° (c 0.270, DMSO). ESI MS: 470([M+H]⁺). HR ESI MS: calcd for C₂₀H₃₆O₆N₇ 470.27216; found 470.27208.

Synthesis of isopropyl2-(2-(2-acetamido-3-methylbutanamido)-5-ureidopentanamido)-6-diazo-5-oxohexanoate(57) Isopropyl2-(2-(2-acetamido-3-methylbutanamido)-5-ureidopentanamido)-6-diazo-5-oxohexanoate(57)

Compound 56 (50 mg, 0.107 mmol), was dissolved in dry DMF (1 mL) and tothis solution was added diisopropylethyl amine (95 μL, 0.543 mmol, 5.1equiv.) was added dropwise followed by acetanhydride (50 μL, 0.532 mmol,5.0 equiv.). The reaction mixture was stirred at room temperature for 2h. The organic solvent was evaporated in vacuo. The residue waschromatographed on silica gel (chloroform:methanol, 7:1) to afford thedesired product 57 (50 mg, 92%) as a yellow amorphous solid. ¹H NMR (400MHz, CDCl₃): 0.82 (3H, d, J=6.9), 0.84 (3H, d, J=7.1), 1.16 (3H, d,J=4.8), 1.17 (3H, d, J=4.9), 1.31-1.53 (3H, m), 1.60-1.68 (1H, m),1.73-1.82 (1H, m), 1.86 (3H, s), 1.89-2.00 (2H, m), 2.31-2.44 (2H, m),2.93-2.97 (2H, m), 4.12-4.17 (2H, m), 4.21-4.26 (1H, m), 4.87 (1H, hept,J=6.3), 5.39 (2H, s), 5.92 (1H, t, J=5.8), 6.02 (1H, s), 7.86 (1H, d,J=8.7), 7.97 (1H, d, J=7.6), 8.20 (1H, d, J=7.5). ¹³C NMR (101 MHz,CDCl₃): 18.20, 19.21, 21.45, 21.49, 22.51, 25.95, 26.56, 29.36, 30.40,36.10, 38.77, 51.33, 52.11, 57.67, 68.01, 158.80, 169.37, 170.97,171.05, 171.70, 194.05. Optical rotation: [α]²² _(D)−22.6° (c 0.257,DMSO). ESI MS: 534 ([M+Na]⁺). HR ESI MS: calcd for C₂₂H₃₇O₇N₇Na534.26467; found 513.26456.

Synthesis of (S)-6-diazo-5-oxo-2-((((4-(((2S,3R,4S,5S,6R)-3, 4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)hexanoate (59)2-(acetoxymethyl)-6-(4-((((6-diazo-1-ethoxy-1,5-dioxohexan-2-yl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (58)

4-[[[(4-Nitrophenoxy)carbonyl]oxy]methyl]phenyl-β-D-glucopyranoside,2,3,4,6-tetraacetate (prepared using a method analogous to the onereported in Angew. Chem. Int. Ed. 2006, 45, 5345-5348, 800 mg, 1.3mmol), was dissolved in dry DMF (6 mL) and a solution of the compound 20(330 mg, 1.66 mmol, 1.3 equiv.), in dry DMF (3 mL) was added dropwise.To this reaction mixture was diisopropylethyl amine (0.91 mL, 5.2 mmol,4 equiv.) was added dropwise. The reaction mixture was stirred at roomtemperature overnight. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform:acetone, 7:1) toeffort the desired product 58 (596 mg, 68%) as a yellow amorphous solid.¹H NMR (400 MHz, CDCl₃): 1.25 (3H, m), 1.93-2.04 (1H, m), 2.02 (3H, s),2.03 (3H, s), 2.04 (3H, s), 2.06 (3H, s), 2.15-2.23 (1H, m), 2.28-2.51(2H, m), 3.85 (1H, ddd, J=10.0, 5.3, 2.5), 4.13-4.20 (3H, m), 4.25-4.34(2H, m), 5.03-5.07 (3H, m), 5.13-5.17 (1H, m), 5.21-5.31 (3H, m), 5.49(1H, d, J=7.9), 6.94-6.97 (2H, m), 7.27-7.31 (2H, m). ¹³C NMR (101 MHz,CDCl₃): 14.24, 20.71, 20.72, 20.80, 21.82, 27.59, 36.50, 53.61, 54.87,61.80, 62.01, 66.56, 68.34, 71.23, 72.16, 72.78, 99.13, 117.05 (2C),129.95 (2C), 131.38, 156.06, 156.86, 169.39, 169.50, 170.32, 170.66,171.91, 193.55. IR (CHCl3): 3429 w, 2110 s, 1757 vs, 1744 sh, vs, 1720sh, s, 1641 m, 1613 m, 1592 w, 1512 s, 1377 m, 1368 s, 1178 m, 1070 sh,s, 651 w cm⁻¹. Optical rotation: [α]²² _(D)−3.3° (c 0.631, CH₂C12). ESIMS: 680 ([M+14]⁺). HR ESI MS: calcd for C₃₀H₃₈O₁₅N₃ 680.22974; found680.22998.

Ethyl(S)-6-diazo-5-oxo-2-((((4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)hexanoate (59)

Compound 58 (833 mg, 1.3 mmol), was dissolved in methanol (20 mL) and ahydrazine hydrate solution 50-60% in water (417 μL, 7.36 mmol, 6.0equiv.) was added dropwise. The reaction mixture was stirred at roomtemperature overnight. The organic solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel (chloroform—methanol 7:1) toeffort the desired product 59 (240 mg, 37%) as yellow amorphous solid.¹H NMR (400 MHz, DMSO): 1.18 (3H, t, J=7.1), 1.72-1.81 (1H, m),1.91-2.00 (1H, m), 2.34-2.45 (2H, m), 3.12-3.19 (1H, m), 3.20-3.28 (2H,m), 3.29-3.34 (1H, m), 3.42-3.48 (1H, m), 3.66-3.71 (1H, m), 3.97-4.03(1H, m), 4.05-4.12 (2H, m), 4.54 (1H, t, J=5.8), 4.86 (1H, d, J=7.3)4.96 (2H, s), 5.01 (1H, d, J=5.3), 5.08 (1H, d, J=4.6), 5.30 (1H, d,J=4.8), 6.05 (1H, s), 7.01 (2H, d, J=8.6), 7.28 (2H, d, J=8.6), 7.67(1H, d, J=7.8). ¹³C NMR (101 MHz, DMSO): 14.07, 25.82, 36.30, 53.27,60.59, 60.70, 65.30, 69.72, 73.23, 76.63, 77.04, 100.28, 116.11 (2C),129.49 (2C), 130.09, 156.16, 157.14, 172.06, 201.32. IR (KBr): 3413 m,2979 w, 2935 w, 2108 m, 1718 m, 1649 m, 1614 m, 1592 w, 1513 m, 1392 m,sh, 1383 m, 1346 m, 1233 s, 1179 m, sh, 1074 s, 1046 s, sh, 1018 m, 948w, sh, 832 w, 511 w cm⁻¹. Optical rotation: [α]²² _(D)−18.9° (c 0.254,DMSO). ESI MS: 534 ([M+Na]⁺). HR ESI MS: calcd for C₂₂H₂₉O₁₁N₃Na534.16943; found 534.16951.

Synthesis of isopropyl2-(2-acetamido-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (60)Isopropyl2-(2-acetamido-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (60)

Compound 38 (425 mg, 1.06 mmol, 1 eq.) was dissolved in dry DMF (8 mL).Pyridine (168 mg, 171 μL, 2.13 mmol, 2 eq.), followed by acetanhydride(130 mg, 121 μL, 1.28 mmol, 1.2 eq.) were added by syringe. The reactionmixture was stirred for 2 h at rt under inert atmosphere. DMF wasevaporated. The crude product was purified by column LC (CHCl₃:MeOH,20:1) to give 423 mg of product 60 as a yellow amorphous solid (90%yield). ¹H NMR (400 MHz, CDCl₃): 1.20 (3H, d, J=6.3), 1.24 (3H, d,J=6.3), 1.81-1.94 (1H, m), 1.98 (3H, s), 2.04-2.33 (3H, m), 3.17 (1H,dd, J=14.6, 7.2), 3.33 (1H, dd, J=14.7, 5.4), 4.37 (1H, td, J=7.7, 4.4),4.75 (1H, td, J=7.4, 5.4), 4.95 (1H, hept, J=6.3), 5.16 (1H, bs), 6.22(d, J=7.6), 6.64 (1H, d, J=7.3), 7.09-7.14 (2H, m), 7.18 (1H, ddd,J=8.2, 7.0, 1.3), 7.32-7.38 (1H, m), 7.66 (1H, dd, J=7.9, 1.1), 8.31(1H, bs). ¹³C NMR (101 MHz, CDCl₃): 21.77, 21.82, 23.38, 26.97, 28.29,36.25, 52.28, 54.07, 54.95, 69.54, 110.38, 111.42, 118.78, 119.79,122.27, 123.57, 127.74, 136.35, 170.25, 170.85, 171.58, 194.04. Opticalrotation: [α]²² _(D)−11.6° (c 0.284, CHCl3). IR (CHCl3):3478 m, 3417 m,3329 w, vbr, 3117 w, 2986 m, 2934 w, 2874 vw, 2855 w, 2110 s, 1732 s,1660 vs, br, 1635 s, sh, 1600 w, sh, 1554 m, br, sh, 1505 s, br, 1467 w,1457 m, 1385 s, sh, 1377 w, vbr, 1350 m, 1183 m, 1146 m, 1105 s, 1093 m,sh, 1012 w. ESI MS: 464 ([M+Na]⁺). HR ESI MS: calcd for C₂₂H₂₇O₅N₅Na464.19044; found 464.19050.

Ethyl 5-(diazomethyl)-3,4-dihydro-2H-pyrrole-2-carboxylate (9a)

Referring to scheme 9, compound 20 (117 mg, 0.588 mmol, 1 eq.) wasdissolved in CH₃CN (1 mL). The reaction mixture was heated for 2 h at60° C. under inert atmosphere. The CH₃CN was evaporated and the crudeproduct was purified by HPLC (CH₃CN/H₂O, HCOOH) affording a light orangeoil (53 mg) in 50% yield. ¹H NMR (CDCl₃): 1.27 (3H, t, J=7.1), 2.83-2.93(1H, m), 2.93-3.07 (2H, m), 3.09-3.20 (1H, m), 4.24 (2H, dq, J=7.1,2.4), 5.17 (1H, dd, J=9.2, 3.5), 7.43 (1H, s); ¹³C NMR (101 MHz, CDCl₃):14.11, 19.98, 34.19, 59.06, 62.53, 126.82, 142.77, 168.70; IR (CHCl₃):2942 w, 2910 w, 2875 vw, 2103 vw, 1747 vs, 1475 w, 1676 m, 1605 vw, 1552w, 1462 w, 1446 w, 1396 w, 1377 m, 1202 vs, 1177 m, 1116 w, 1095 m,cm⁻¹; ESI MS: 182 ([M+H]⁺); HR ESI MS: calcd for C₈H₁₂O₂N₃ 182.0930;found 182.0931.

Isopropyl 5-(diazomethyl)-3,4-dihydro-2H-pyrrole-2-carboxylate (9b)

Referring to scheme 9, compound 3 (100 mg, 0.469 mmol, 1 eq.) wasdissolved in CDCl₃ (2 mL). The reaction mixture was stirred at rtovernight. The CDCl₃ was evaporated and the crude product was purifiedby HPLC (CH₃CN/H₂O, HCOOH) and a light orange oil (41 mg) was obtainedin 45% yield. ¹H NMR (CDCl₃): 1.25 (3H, d, J=6.1), 1.27 (3H, d, J=6.1),2.81-2.95 (2H, m), 2.96-3.06 (1H, m), 3.08-3.18 (1H, m), 5.09 (1H, hept,J=6.1), 5.13 (1H, dd, J=9.1, 3.4), 7.43 (1H, s); ¹³C NMR (101 MHz,CDCl₃): 20.00, 21.69 (2C), 21.75, 34.24, 59.20, 70.48, 126.84, 142.71,168.25; Optical rotation: [α]²² _(D)−31.8° (c 0.110, CHCl₃); IR (CHCl₃):2104 w, 1741 vs, 1675 w, 1644 w, 1551 w, 1465 m, 1388 m, sh, 1376 m,1209 s, 1182 m, 1147 m, 1106 s, cm⁻¹; ESI MS: 196 ([M+H]⁺); HR ESI MS:calcd for C₉H₁₄O₂N₃ 196.10805; found 196.10808.

Example 5 Evaluation of DON Prodrugs

Overview.

The presently disclosed subject matter demonstrates profound efficacy ofDON in murine model of GBM, although overt toxicities were observed. Inattempt to increase DON's therapeutic index, several DON prodrugs weresystematically synthesized. The initial strategy involved masking DON'scarboxylic acid with simple alkyl esters, such as ethyl ester 20 andisopropyl ester 3 (FIG. 20). However, 3 and 20 exhibited chemicalinstability cyclizing to form a unique diazo-imine. Given thisinstability, both the primary amine and the carboxylate of DON were nextmasked with prodrug moieties. Three types of amine promoieties wereused, including (oxodioxolenyl)methyl carbamate esters (13 and 36),dipeptides (9 and 25), and pivaloyl-oxy-methyl (POM)-based esters (14,32 and 42). The dual promoiety-containing prodrugs resulted insufficient chemical stability permitting further evaluation in in vitrometabolic stability assays. While all of the prodrugs exhibited rapidmetabolism in mouse plasma, some provided excellent plasma stability inmonkeys and humans. When evaluated in vivo, the most stable DON prodrug(5c, methyl-POM-DON-isopropyl-ester) achieved 10-fold enhancedbrain:plasma ratio versus DON in monkeys, thus providing a possibleclinical path to DON utilization in GBM patients.

Chemistry.

Scheme 9 outlines the synthesis and characterization of the ester basedprodrugs 20 and 3 of DON and their subsequent cyclization to noveldiazo-imines 9a-b. The pyroglutamate esters 6a-b (D'Andrea, et al.,2008) were FMOC-protected to afford compounds 18 and 1. Formation of thediazoketones 19 and 2 was accomplished using TMS diazomethane in goodyield. Rapid deprotection with piperidine afforded the ester basedprodrugs 20 and 3. Unexpectedly slow cyclization was observed even undermild conditions (e.g. stirring in chloroform at room temperature)affording the novel diazo-imines 9a and 9b. It is believed that this isthe first example of this functional group being described in thechemical literature. Attempts to avoid the cyclization of 20 and 3 bysalt formation to protonate the amine resulted in instability of thediazo group. Furthermore, unlike most imines, the 5-member cyclicdiazo-imines 9a-b were found to be stable, even at acidic pH, and didnot convert back to DON esters (results not shown).

TABLE 3 Novel Diazo-imines 9a

9b

Given their sufficient chemical stability, compounds 20 and 3 wereutilized as synthetic intermediates for preparation of dual promoietyprodrugs (Schemes 8 and 2). The (oxodioxolenyl) methyl carbamate adducts36 and 13 were synthesized as outlined in Schemes 8 and 2. The4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one was reacted with S-ethylcarbonochloridothionate, (Keicher, et al., 2009) followed byN-hydroxysuccinimide to provide 12. Reaction of 20 and 3 with 12afforded the (oxodioxolenyl) methyl carbamate esters 36 and 13 inexcellent yields.

Schemes 1, 4, and 7 outline the synthesis of DON dipeptide esters. DONesters 20 and 3 were coupled with Fmoc-L-leucine or Fmoc-L-tryptophanusing HBTU in high yield to form the protected dipeptides 33 and 37(scheme 7). Deprotection with diethyl amine or piperidine afforded thedesired leucine-DON 25 (scheme 4) and 9 (scheme 1) and tryptophan-DONprodrugs 34 and 38 (scheme 7).

As shown in Scheme 6, the POM derivative 42 was synthesized from 20using the POM-N-hydroxysuccinimate ester (Gallop, et al., 2008) in 40%yield. Introduction of methyl group in the POM ester led to theformation of methyl-POM derivatives 32 (scheme 6) and 14 (scheme 2),with an added chiral center. Both 32 and 14 were synthesized from 20 and3 using 1-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl pivalate(Gallop, et al., 2008). 32 was obtained as a mixture of twodiastereomers (1:1 ratio) in 68% yield. 14 was also obtaineddiastereomeric mixture (1:1) ratio, but was further purified into itscorresponding diastereomers 14a and 14b, of which diastereomer 14b wasused for subsequent biological testing as described below. The acetalmethyl stereochemistry of 14a and 14b was assigned arbitrarily.

Results and Discussion.

DON showed robust inhibition of glutamine metabolism and antitumorefficacy in a murine GBM model. Despite several lines of evidenceindicating the potential therapeutic efficacy of targeting glutaminemetabolism in GBM, the effect of DON on GBM tumor growth has not yetbeen reported in vivo. Using the U87 flank xenograft mouse model of GBM,(Eshleman, et al., 2002) it has been first confirmed that systemicadministration of DON (0.8 mg/kg, i.p) inhibited glutamine metabolism asreflected by an accumulation of endogenous glutamine in the tumor (FIG.21A; p<0.05) similar to other model systems. (Willis, et al., 1977;Windmueller, et al., 1974) Then its antitumor efficacy was evaluated,and observed that DON not only halted tumor growth, but also effectivelyinduced tumor regression. Specifically, vehicle-treated mice displayedsignificant tumor growth over the course of the experiment, whileDON-treated mice (0.8 mg/kg, i.p, q.d.) exhibited >50% reduction intumor volume (FIG. 21B; main effect of time [F(3,48)=6.049, p=0.0014];treatment [F(1,16)=33.42, p<0.0001]; interaction [F(3,48)=21.70,p<0.0001]). Although DON exhibited excellent anti-tumor efficacy, allmice receiving DON displayed significant signs of toxicity includingweight loss (12±4.1%), hunching, ptosis, and lethargy. These findingsare consistent with other reports of DON's efficacy and toxicity both invitro and in vivo. (Fogal, et al., 2015; Cervantes-Madrid, et al., 2015;Potter, et al., 2015)

Some simple DON alkyl ester prodrugs found to be unstable. Masking bothDON's carboxylate and amine functionalities enhanced the stabililty ofcertain prodrugs. A prodrug strategy is often employed to enhance tissuepenetration and change the pharmacokinetic parameters of effectivedrugs. Indeed, prodrug strategies are common in drug development as 5-7%of the approved worldwide drugs are prodrugs (Rautio, et al., 2008). Theinitial prodrug strategy for DON involved masking the carboxylic acidwith simple alkyl esters such as ethyl 20 and isopropyl 3. The synthesisof these two derivatives was straightforward affording compounds 20 and3 in good yield. It is surprising that these simple DON alkyl esters hadnot previously been reported in the chemical literature, given that DONchemistry and utility has been described by numerous groups for over 60years (Magill, et al., 1957; Dion, et al., 1956; Magill, et al., 1956;Coffey, et al., 1956). One potential reason is that it has beendiscovered that 20 and 3 were unstable, slowing cyclizing to form uniquediazo-imines 9a and 9b. These two unique derivatives were found to bechemically stable even at acidic pH, precluding their use as DONprodrugs.

Given the instability of certain simple ester prodrugs, both the primaryamine and the carboxylate of DON were masked with prodrug moieties. Thisdual promoiety strategy was rationalized to eliminate the potential forcyclization and potentially further improve the lipophilicity. Threeamine promoieties including (oxodioxolenyl)methyl carbamate esters wereused (FIG. 20, compounds 36, 13), dipeptides (25 and 38), andpivaloyl-oxy-methyl (POM)-based esters (42, 32, 14b). These promoeitieswere chosen because they target distinct metabolic enzymes includingparaoxonase, aminopeptidases, and carboxylesterases, respectively. Toimpart further metabolic stability of the POM derivative (Table 3, 42),corresponding methyl-POM analogs were prepared (32, 14b). All dualpromoiety-containing prodrugs exhibited sufficient chemical stability topermit further evaluation.

All DON prodrugs were rapidly metabolized in mouse plasma, however 32and 14b found to be stable in human and monkey plasma. Table 3 outlinesthe plasma stability of DON prodrugs 36, 13, 25, 9, 34, 38, 42, 32 and14b. All prodrugs were completely metabolized in mouse plasma within the60 min incubation time. However in monkey and human plasma, the prodrugs32 and 14b, with methyl-POM on the amine and ethyl or isopropyl ester onthe carboxylate respectively, demonstrated moderate/high stability with60-75% of the prodrug remaining in monkey plasma, and 80-90% remainingin human plasma within the 60 min incubation time. Given 14b had thebest stability profile in human plasma, it was selected for furtherevaluation in pharmacokinetic studies and compared to DON for itsability to penetrate the brain and liberate DON.

TABLE 4 PLASMA STABILITY Compound # Mouse Monkey Human 36 0 0 0 13 0 0 025 0 1 1  9 0 1 1 34 0 4 12 38 0 10 30 42 0 0 9 32 0 75 88 14b 0 61 91

Lead prodrug 14 enhanced brain delivery of DON in monkeys but not inmice.

As expected from a DON prodrug which is completely metabolized in mouseplasma, it has beenfound that oral administration of DON (1) (0.8 mg/kg)and 14b (0.8 mg/kg equivalent) exhibited similar DON plasma (FIG. 22A)and brain (FIG. 22B) concentration profiles when dosed in mice. TheAUC_(0-t) of DON following administration of DON and 14b in plasma were1.25 nmol*h/mL and 1.22 nmol*h/mL respectively, suggesting rapid andcomplete liberation of DON from 14b in vivo. Similarly in the mousebrain, the AUC_(0-t) of DON following DON or 14b administration was 0.57nmol*h/g and 0.69 nmol*h/g, respectively, with the brain/plasmaapproximately 0.46 from DON vs 0.56 from prodrug 14b. Thesepharmacokinetic results corroborated the in vitro metabolism studiessuggesting 14b was completely converted to DON in mouse plasma.

Following the mouse studies, the pharmacokinetics of DON and 14b wereevaluated in monkeys, as monkeys better mimicked the human plasmastability profile. In pigtail macaques, i.v. administration of DON and14b (1.6 mg/kg DON equivalent) demonstrated significantly different DONplasma profiles FIG. 23A). DON administration provided high plasmaexposures with AUC_(0-t) of 42.7 nmol*h/mL. In contrast, 14badministration delivered about 7 fold lower plasma exposure of DON withAUC_(0-t) of 5.71 nmol*h/mL. The opposite observation was seen in theCSF where enhanced DON levels were observed after 14 administration. Inthe CSF at 30 min post dose, DON administration resulted in 0.33 nmol/gDON while 14b delivered 1.43 nmol/g DON. When comparing plasma to CSFratio at 30 min, 14b demonstrated 10-fold enhancement of DON CSFdelivery versus DON (FIG. 23B).

The glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON, 1) has shownrobust anti-cancer efficacy in preclinical and clinical studies, but itsdevelopment was halted due to marked systemic toxicities. Herein it hasbeen demonstrated that DON inhibits glutamine metabolism and providesantitumor efficacy in a murine model of glioblastoma, although toxicitywas observed. To enhance DON's therapeutic index, a prodrug strategy wasused to increase its brain delivery and limit systemic exposure.Unexpectedly, simple alkyl ester-based prodrugs were ineffective due tochemical instability cyclizing to form a unique diazo-imine. However,masking both DON's amine and carboxylate functionalities impartedsufficient chemical stability for biological testing. While these dualmoiety prodrugs exhibited rapid metabolism in mouse plasma, severalprovided excellent stability in monkey and human plasma. The most stablecompound (14b, methyl-POM-DON-isopropyl-ester) is highly stable inplasma from monkey, human, pigs and dog but is rapidly metabolized inmice. When evaluated in monkeys at an equimolardoses, 14b deliveredlower levels of DON to plasma but higher levels in CSF it achieved10-fold enhanced brain:plasma ratio versus DON. This strategy mayprovide a path to DON utilization in GBM patients.

Example 6 Compound 14b Enhanced CSF Delivery of DON in Monkey

Method

Compound:

Compound 14b was dissolved in 50 mM HEPES buffered saline containing 5%ethanol and 5% tween on the date of administration.

Monkey:

Monkey studies were conducted according to protocol (#PR15M298) approvedby the Animal Care and Use Committee at Johns Hopkins University. Twofemale pigtail monkeys (approximately 3.5 kg, non-drug naive) wereadjacently housed in stainless steel cages on a social interaction rack(contains 4 cages, each 32.5″ wide×28″ deep×32″ high) maintainingtemperature of 64-84° F., humidity of 30-70% with alternating 14-10 hourlight/dark cycle as per the USDA Animal Welfare Act (9 CFR, Parts 1, 2,and 3). Food was provided daily in amounts appropriate for the size andage of the animals and RO purified water provided ad libitum through anin-cage lixit valve. Food enrichment was provided Monday through Friday.

Treatment:

Prior to drug administration, macaques were sedated with ketamine givenas an intramuscular injection prior to test article administration.Sedation was maintained through blood and cerebrospinal fluid (CSF)sample collections with ketamine at a starting rate of 15 mg/kg withadditional doses of 20-30 mg during the first hour. At subsequent timepoints ketamine was given at 10-15 mg/kg. DON (50 mM HEPES bufferedsaline) and compound 14b (50 mM HEPES buffered saline containing 5%ethanol and 5% tween) were administered (1.6 and 3.6 mg/kg equivalentdose of DON) to the animals at a dosing volume of 1 mL/kg intravenously.CSF sample (target of 50 μL) was obtained by percutaneous puncture ofthe cisterna magna at 30 min post dose. Blood samples (1 mL) werecollected at 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h post dose bypercutaneous puncture of a peripheral vein. Samples were processed forplasma (centrifuged at a temperature of 4° C., at 3,000 g, for 10minutes). All samples were maintained chilled on ice throughoutprocessing. Samples were collected in microcentrifuge tubes, flashfrozen, and placed in a freezer set to maintain −80° C. until LC/MSanalysis.

Data Analysis:

DON was extracted from samples (50 mg) with 250 μL methanol containingglutamate-d₅ (10 μM ISTD) by vortexing in low retention tubes. Sampleswere centrifuged at 16,000 g for 5 minutes to precipitate proteins.Supernatants (2004) were moved to new tubez and dried at 45° C. undervacuum for 1 hour. To each tube, 50 μL of 0.2 M sodium bicarbonatebuffer (pH 9.0) and 100 μL of 10 mM dabsyl chloride in acetone wasadded. After vortexing, samples were incubated at 60° C. for 15 minutesto derivatize. Samples (2 μL) were injected and separated on an Agilent1290 equipped with an Agilent Eclipse plus C18 RRHD 2.1 X100 mm columnover a 2.5 minute gradient from 20-95% acetonitrile+0.1% formic acid andquantified on an Agilent 6520 QTOF mass spectrometer. Calibration curvesover the range of 0.005-17.1 μg/mL in plasma and CSF for DON wereconstructed from the peak area ratio of the analyte to the internalstandard using linear regression with a weighting factor of 1/(nominalconcentration). Correlation coefficient of greater than 0.99 wasobtained in all analytical runs. The mean predicted relative standarddeviation for back calculated concentrations of the standards and QC'sfor all analytes were within the range of 85 to 115%, except for thelowest concentration which was within the range of 80 to 120% with anoverall accuracy and precision of 6.7% and 6.6% respectively.

Results

The pharmacokinetics of DON and compound 14b in monkeys were evaluated.In pigtail macaques, i.v. administration of DON (1.6 mg/kg) and compound14b (3.6 mg/kg; 1.6 mg/kg DON equivalent) demonstrated significantlydifferent DON plasma profiles (FIG. 58A). DON administration providedhigh plasma exposures with AUC0-t of 42.7 nmol*h/mL. In contrast,compound 14b administration delivered ˜7 fold lower plasma exposure ofDON with AUC0-t of 5.71 nmol*h/mL. The opposite observation was seen inthe CSF where enhanced DON levels were observed after compound 14badministration. In the CSF at 30 min post dose, DON administrationresulted in 0.33 nmol/g DON while compound 14b delivered 1.43 nmol/gDON. When comparing plasma to CSF ratio at 30 min, compound 14bdemonstrated unexpected 10-fold enhancement of DON CSF delivery versusDON (FIG. 58B).

Example 7 Compounds 14b and 47 Enhanced CSF Delivery of DON in Swine

Method

Compound:

Compound 47 was dissolved in a sterile saline containing 5% ethanol and5% Tween 80 on the date of administration.

Swine:

Swine studies were conducted under a protocol approved by the JohnsHopkins Animal Care and Use Committee. Adult, female Gôttingen×Yucutanminiature swine (Massachusetts General Hospital, MA) were housed inJohns Hopkins University facilities accredited by the Association forAssessment and Accreditation of Laboratory Animal Care International incompliance with the Animal Welfare Act, Animal Welfare Regulations, andthe Public Health Service Policy on the Humane Care and Use ofLaboratory Animals. Animals were maintained on a 14-h light and 10-hdark schedule, provided ad libitum water and a commercial miniswine diet(Teklad, Madison, Wis.) with environmental enrichment (fruit/vegetables)twice daily.

DON and Compounds 14b and 47 Treatment:

Animals were individually housed while on study in order to monitorbehavior and clinical health following drug administration. Whole bloodfor drug pharmacokinetic evaluation was collected from a dual lumencentral venous catheter (CVC) implanted in the external jugular veinprior to study initiation. Animals were anesthetized with a combinationof ketamine hydrochloride (20-30 mg/kg, i.m.) and xylazine (2 mg/kg,i.m.), intubated, and maintained under isoflurane (1-2%) inhalantanesthesia. A temporary peripheral saphenous vein catheter was placed inthe hind limb to allow for anatomical separation of drug infusion andwhole blood sampling via CVC. DON and compounds 14b and 47 weredissolved in a sterile saline solution containing 5% ethanol and 5%Tween 80 prior to i.v. infusion via saphenous vein catheter over 1 hour(1 ml/min) for a final dose of 1.6 mg/kg or molar equivalentadministered at 1 ml/kg (n=1/dose). Blood samples (1 mL) were taken fromCVC at predose, 5, 15, 30, 45, and 60 min. Plasma was separated by lowspeed centrifugation at 3000 g for 10 min at 4° C. CSF was obtained fromthe cisterna magna using a 3.5 in×22 gauge spinal needle (BectonDickinson Health Care, Franklin Lakes, N.J., USA) at 60 min post-dose.All samples were flash frozen upon harvest and stored at −80 C untilbioanalysis.

Data Analysis:

Quantitation of DON in plasma, CSF, and brain homogenate by LC-MS/MS wasperformed. Briefly, DON was extracted from plasma, CSF, and brainsamples with methanol containing glutamate-d₅ (10 μM ISTD) by vortexingfollowed by centrifugation 16000 g for 5 min. Supernatants werealiquoted and dried at 45° C. for under vacuum for 1 h. Sodiumbicarbonate buffer (0.2M, pH 9.0) and dabsyl chloride (10 mM) in acetonewere added to each tube, mixed, and incubated for 15 min at 60° C. toderivatize. Samples were then injected and separated on an Agilent 1290equipped with an Agilent Eclipse plus C18 RRHD 2.1×100 mm column over a2.5 min gradient from 20 to 95% acetonitrile+0.1% formic acid andquantified on an Agilent 6520 QTOF mass spectrometer. Peak area ratio ofthe analyte to the internal standard was plotted against a 14 standardcurve to yield DON concentrations for each sample.

Result

The pharmacokinetic of DON, compound 14b and compound 47 were evaluatedin swine. IV administration of compounds 14b and 47 (1.6 mg/kg DONequivalent dose) resulted in 3-5-fold lower DON plasma exposuresrelative to an equimolar dose of DON (FIG. 59A). Plasma AUC_(0-t) forDON and compounds 14b and 47 were 29.9, 8.00 and 5.70 nmol·h/mL,respectively. The opposite trend occurred in CSF, where compounds 14band 47 delivered substantially higher amounts of DON to the CSF (FIG.593B), resulting in unexpected increased CSF-to-plasma ratios (FIG.59C).

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art. In case of aconflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

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Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A compound selected from the group consistingof:


2. The compound of claim 1 which is:


3. A pharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier, diluent, or excipient.
 4. Apharmaceutical composition comprising the compound of claim 2 and apharmaceutically acceptable carrier, diluent, or excipient.